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PubMed1983

Effects of trimethoprim-sulfamethoxazole and incubation atmosphere on isolation of group A streptococci.

The effects of selective media and incubation atmosphere on the isolation of group A beta-hemolytic streptococci were evaluated. A higher percentage of group A streptococci was isolated on sheep blood agar incubated in air than in CO2 or anaerobic atmospheric conditions. Fewer non-group A beta-hemolytic streptococci were isolated on sheep blood agar incubated in air than in CO2 or anaerobically. Group A streptococcal isolation was not significantly affected by different incubation atmospheres on sheep blood agar containing trimethoprim-sulfamethoxazole, but detection time was longer than on sheep blood agar alone. No significant difference was found between isolation of group A streptococci on sheep blood agar incubated in air and that on sheep blood agar containing trimethoprim-sulfamethoxazole and incubated in 5 to 10% CO2; however, more group A streptococci were isolated on sheep blood agar in air within 24 h. Sheep blood agar incubated at 35 degrees C in air is, therefore, recommended for the isolation of group A streptococci from throat swabs.

Air↗
PubMed1992

A new approach to estimating AIDS incubation times: results in homosexual infected men.

STUDY OBJECTIVE: The aim was to develop a new approach for estimating the incubation period of acquired immunodeficiency syndrome (AIDS), based on age distributions. DESIGN: Incubation period was expressed as the difference between age at time of diagnosis and age at time of contamination. Assuming independence between age at time of infection and incubation period, the age distribution of newly diagnosed AIDS cases is thus the convolution product between the distributions of the age of freshly infected patients and of the incubation times. AIDS incubation time can therefore be estimated from the age distribution of newly HIV infected subjects and newly diagnosed AIDS cases. SUBJECTS: Subjects were 2220 AIDS cases diagnosed until 1987, reported to the Ministry of Health, France, and 172 subjects discovered to be HIV-1 seropositive during a blood donation in Paris between August 1985 and July 1988. In both groups, the only known risk factor was homosexuality. MAIN RESULTS: The estimated median incubation time was 9.9 years (90% CI 9.0-10.9 years). Confidence intervals were narrow, even when taking into account the uncertainty in serodetection delay (90% CI 6.7-13.5 years). CONCLUSIONS: The incubation estimate is as accurate as previous estimates based on other models. This technique could therefore be applied to other risk groups.

Acquired Immunodeficiency Syndrome↗
PubMed1982

Effects of incubation at various 'room' temperatures on the sensitivity and specificity of Microstix-3.

The feasibility of room temperature incubation of Microstix-3 specimens to detect urinary tract infection was studied. This procedure, if successful, would allow home monitoring of patients and eliminate the need for an incubator. Four Microstix-3 strips were dipped into each of 1,241 urine specimens and incubated at 18, 22, 27 or 37 degree C. Results after incubation for 24 h at 27 degree C and after 48 h at 22 degree C were comparable to those after incubation for 18-24 h at 37 degree C. However, even after 72 h, incubation at 18 degree C did not produce results comparable to those obtained at 37 degree C. It appears that the incubator can be successfully eliminated at the higher temperatures tested if the limits of time outlined are observed.

Bacteriuria↗
PubMed1983

Fc gamma receptors on lymphocytes from normal donors and patients with lymphoproliferative diseases. Influence of incubation conditions.

The antibody-coated human erythrocyte and the antibody-coated ox erythrocyte rosette assays (EAHu and EAOx) were compared to detect Fc gamma receptors on human peripheral blood lymphocytes. Two incubation conditions were examined: 1 h at room temperature and overnight at 4 degrees C. In healthy persons, in patients with Hodgkin's disease and in patients with non-Hodgkin lymphoma (NHL) the mean percentage of EAHu-rosette-forming cells (EAHu-RFC) increased significantly when the incubation was carried out overnight at 4 degrees C instead of 1 h at room temperature. This increase was caused by Fc gamma receptor-bearing T cells. In the case of EAOx-RFC only a slight increase was found. The percentage of EAHu-RFC and EAOx-RFC differed significantly in the healthy group after the overnight incubation and in the NHL group after the 1-hour incubation. When comparing the mean percentage of EA-RFC in the patient groups with that of the healthy persons significant increases were observed: EAHu-RFC in patients with Hodgkin's disease in the overnight incubation and EAOx-RFC in patients with Hodgkin's disease and NHL in both incubation conditions. In patients with chronic lymphocytic leukemia (CLL, B-cell type) the mean percentage of EAHu-RFC was very low, however that of EAOx-RFC was moderate to high. It is concluded that in the two rosette assays the antigen-antibody complexes may have different avidities to different lymphocyte subpopulations, and that incubation conditions may have an influence on this avidity.

Animals↗
PubMed1986

Correlation between pyridoxal-5'-phosphate levels and the percentage activation of aspartate aminotransferase enzyme in haemolysate and plasma during in vitro incubation studies with different B6 vitamers.

Conflicting results using erythrocyte aminotransferase (eAST) stimulation to assess vitamin B6 nutritional status in patients with less severe B6 deficiencies are common. It has been claimed that the presence of different B6 vitamers may modify the activation of eAST by pyridoxal-5'-phosphate (PLP) leading to stimulatory or even inhibitory effects. To investigate the possible role of this phenomenon in producing inconsistent AST stimulations, aliquots of whole blood were incubated with equivalent amounts of different B6 vitamers, and the AST stimulation was correlated with the concentrations of PLP, measured by high-performance liquid chromatography. At the end of the incubation period the erythrocytes and plasma were separately analyzed. The conversion of non-PLP B6 vitamers to PLP, by the erythrocytes, was similar (approximately 70%) for all B6 vitamers used in the incubation experiments. The newly formed PLP accumulated in the erythrocytes, but the percentage activation of AST did not change significantly from the basal levels, in spite of the presence of increased levels of PLP and other B6 vitamers used for incubation. When PLP was used in the incubation studies, all of it was retained by the plasma and was associated with a marked suppression of plasma AST stimulation. To determine the degree to which plasma and erythrocyte AST was dose-dependent, plasma and haemolysates were incubated with increasing concentrations of PLP. A very significant inverse relationship was obtained in plasma between AST stimulation and PLP even at modest PLP levels, while haemolysates required incubation with much higher PLP concentrations to demonstrate the same effect. Since plasma PLP is considered to be the most reliable indicator of B6 nutritional status in man, our findings suggest that plasma percentage AST stimulation more closely reflects the B6 nutritional status than erythrocyte AST stimulation test which may reflect B6 status only in severe, longstanding B6 deficiencies. Conflicting results using erythrocyte AST stimulations may be attributed to the insensitivity of red cell AST to changes in PLP content. It is unlikely that the presence of non-PLP B6 vitamers in haemolysate may affect the percentage stimulation of aminotransferase enzymes by PLP.

Aspartate Aminotransferases↗
PubMed1999

Hydrolytic and enzymatic incubation of polyhydroxyoctanoate (PHO): a short-term in vitro study of a degradable bacterial polyester.

The present study examined the degradation behaviour of poly(beta-hydroxy octanoate) (PHO), a bacterial poly(beta-hydroxy alkanoate), following incubation under hydrolytic or enzymatic conditions in vitro. Solution-cast PHO films were incubated in a citrate buffer solution with and without acid phosphatase and in an acetate buffer with and without beta-glucuronidase for periods ranging from 7 to 60 days. The physical characterization of the PHO films was analyzed by SEM and tensile strength studies. In addition, various analytical methods were used to detect modifications in the chemical and morphological structure of the PHO, namely, ESCA, FTIR, DSC, X-ray diffraction, and SEC. The results indicate that the enzymatic conditions selected in the present study induced no significant surface morphological or chemical modifications, and no significant weight loss was observed after 60 days of incubation. However, as revealed by weight average molecular weight Mw and number average molecular weight Mn decreases, changes in the bulk structure of the PHO were observed with acid phosphatase at 28 and 60 days, in contrast to smaller Mw and Mn decreases recorded in both the buffers and the beta-glucuronidase. The tensile properties had decreased following incubation, yet showed no difference under all of the selected conditions. With no weight loss or surface changes, the PHO films incubated in acid phosphatase showed only a chemical hydrolytic process characterized by Mw and Mn decreases with time of incubation. The present study demonstrated that the degradation of PHO films is one of slow, chemical hydrolysis only, perhaps requiring several months of incubation. The hydrophobic nature of the long alkyl pendent chain in PHO may be responsible for this slow process. The inability of enzymes to degrade PHO may be attributed to the latter's poor adsorption capacity, due to its hydrophobic nature, and to a lack of specificity in the catalytic activity of these enzymes.

Acid Phosphatase↗
PubMed2000

Modulation of cholesterol concentration in Caco-2 cells by incubation with different n-6 fatty acids.

Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 microM of either linoleic acid (LA, 18:2n-6), gamma-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 microM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.

Caco-2 Cells↗
PubMedDate not supplied

Pathogenicity of Choristoneura fumiferana nucleopolyhedrovirus propagated in vitro at different incubation temperatures.

To optimize the in vitro production of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) as a potential microbial pest control agent, the pathogenicity of occlusion bodies (OBs) produced in two cell lines at three incubation temperatures was determined by bioassay. A plaque-purified isolate of CfMNPV was amplified in permissive C. fumiferana cell lines, FPMI-CF-203 and FPMI-CF-2C1, and incubated at 22, 24, and 28 degrees C. Occlusion bodies propagated in FPMI-CF-203 cells at 28 degrees C were significantly larger (17.5 microm(3)) and more pathogenic (LD(50) = 27; LD(95) = 185, where LD(50) and LD(95) are doses required to kill 50 and 95% of the test larvae, respectively) than those produced in either of the cell lines at any of the incubation temperatures tested. Increased temperatures yielded larger OBs from both cell lines. The pathogenicity of OBs propagated in the FPMI-CF-203 cell line increased with incubation temperature, whereas that of OBs produced in FPMI-CF-2C1 cells decreased. Comparison of the pathogenicity of OBs, whether naturally occurring or genetically modified, should be standardized by cell line and incubation temperature used for propagation. Production efficiency decreased with increasing incubation temperature for each cell line. Lower incubation temperatures used for propagation, and standardization of the titer of viral inoculum, should be further investigated to determine the economic feasibility of the in vitro production of CfMNPV as a microbial pest control agent.

Animals↗
PubMed1983

Zymosan-induced chemiluminescence of granulocytes in incubated human whole blood. The role of platelets.

Zymosan-induced chemiluminescence (ZI-CL) of whole blood which is mainly the response of granulocytes can be used as a rapid, simple and reliable diagnostic tool for hematological and allergic disorders. ZI-CL of whole blood remained unchanged when stored at 0 degree C for 4 hr, whereas it increased by incubation at 37 degrees C. This phenomenon points to platelet-granulocyte interaction during the incubation. ZI-CL of freshly prepared suspension of leukocytes and erythrocytes was enhanced by adding platelet rich plasma (PRP) obtained from incubated blood. Enhanced CL response was observed when erythrocytes or factors derived there of were present in incubated blood. Platelet stimulating factor (PSF) was prepared from incubated erythrocyte suspension. This factor also increased ZI-CL of granulocytes when incubated with fresh PRP. The consistent inhibition of CL of incubated blood by apyrase (0.5 U/ml) and the increased response of fresh blood by the addition of ADP (10(-5) M) suggested that this factor might be ADP itself. Platelets stimulated by this factor may respond more markedly to added zymosan for still unexplored reason with enhanced CL.

Adenosine Diphosphate↗
PubMed1980

Incubation in mallards (Anas platyrhynchos): changes in plasma levels of prolactin and luteinizing hormone.

The concentrations of prolactin and LH were measured in the plasma of male and female mallards (Anas platyrhynchos) during the breeding season. The concentration of prolactin in the female birds was low before and during egg-laying, increased threefold (P less than 0.001) during incubation and returned to basal levels immediately after the young were hatched. The drakes, which do not participate in incubation in this species, had fairly low prolactin levels throughout the breeding period; levels being slightly higher during the incubation phase than at other times but still much lower (P less than 0.001) than in the incubating females. Concentrations of LH in the females were high during egg-laying but declined at the onset of incubation. In the drakes levels of LH remained high for much longer and did not appear to decline at a fixed time relative to the reproductive cycle of the female birds. These observations support the view that prolactin is associated with incubation in birds, though the data do not permit a conclusion as to whether an increase in prolactin secretion causes incubation behaviour or is stimulated by it.

Animals↗
PubMed1989

The role of hypothalamic vasoactive intestinal polypeptide in the maintenance of prolactin secretion in incubating bantam hens: observations using passive immunization, radioimmunoassay and immunohistochemistry.

The role of chicken vasoactive intestinal polypeptide (cVIP) as a prolactin-releasing factor was investigated in incubating bantam hens. Specific antibodies were raised against cVIP (anti-cVIP) for passive immunization studies, to develop a radioimmunoassay and to localize VIP neurones immunohistochemically in the hypothalamus. The concentration of plasma prolactin decreased after i.v. injection of anti-cVIP: this low concentration being maintained by daily injection of anti-cVIP. Incubating hens injected daily with anti-cVIP deserted their nests after 4.5 +/- 0.6 days and returned to lay after 20 +/- 1 days. This disruption of incubation behaviour with anti-cVIP was prevented by concomitant, twice daily, injections of 30 IU ovine prolactin. The concentration of plasma LH was not immediately affected after injection of anti-cVIP but increased when the hens deserted their nests. The amount of cVIP, measured by radioimmunoassay, was significantly higher in the median eminence (P less than 0.01) and medial basal hypothalamus (P = 0.05) in incubating than in laying hens. No differences were seen in the amounts of cVIP in the preoptic hypothalamus or in a part of the forebrain including the nucleus accumbens, between laying and incubating hens. Morphological observations were made on immunohistochemically identified cVIP cell bodies in the medial basal hypothalamus. These showed that cVIP cell number, cell area and density of immunoreactive product were significantly (P less than 0.05) greater in incubating than in laying hens. Further, the density of cVIP reaction product in the anterior median eminence was also significantly (P less than 0.01) greater in incubating than in laying hens.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Evidence for a sustained increase in clonal beta-cell basal intracellular Ca2+ levels after incubation in the presence of newly diagnosed Type-1 diabetic patient sera. Possible role in serum-induced inhibition of insulin secretion.

We have previously reported that newly diagnosed Type-1 diabetic patient sera potently suppressed insulin secretion from a clonal rat pancreatic beta-cell line (BRIN BD11) but did not alter cell viability. Here, we report that apoptosis in BRIN BD11 cells incubated in various sera types (fetal calf serum (FCS), normal human serum and Type-1 diabetic patient) was virtually undetectable. Although low levels of necrosis were detected, these were not significantly different between cells incubated in sera from different sources. ATP levels were reduced by approximately 30% while nitrite production increased twofold from BRIN BD11 cells incubated for 24 h in the presence of Type-1 diabetic patient sera compared with normal human sera. Additionally, ATP levels were reduced by approximately 40% and DNA fragmentation increased by more than 20-fold in BRIN BD11 cells incubated in FCS in the presence of a pro-inflammatory cytokine cocktail (interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma), compared with cells incubated in the absence of cytokines. Nitric oxide production from BRIN BD11 cells was markedly increased (up to 10-fold) irrespective of sera type when the cytokine cocktail was included in the incubation medium. Type-1 diabetic patient sera significantly (P<0.001) raised basal levels of intracellular free Ca(2+ )concentration ([Ca(2+)](i)) in BRIN BD11 cells after a 24-h incubation. The alteration in [Ca(2+)](i) concentration was complement dependent, as removal of the early complement components C1q and C3 resulted in a significant reduction (P<0.01) of sera-induced [Ca(2+)](i )changes. We propose that the mechanism of Type-1 diabetic patient sera-induced inhibition of insulin secretion from clonal beta-cells may involve complement-stimulated elevation of [Ca(2+)](i) which attenuates the nutrient-induced insulin secretory process possibly by desensitizing the cell to further changes in Ca(2+).

Adenosine Triphosphate↗
PubMed2003

A salivary incubation test for evaluation of oral malodor: a pilot study.

BACKGROUND: Breath odor is scored by different techniques, each with its own shortcomings. Organoleptic ratings are uncomfortable for the patient, subjective, influenced by external parameters including food and cosmetics, and especially lack international calibration. Portable sulphide monitors are relatively expensive and neglect several major malodorous molecules (e.g., butyric and propionic acids, putrescine, and cadaverine). Gas chromatography necessitates expensive devices and experienced technicians. This pilot study explored the applicability of a new technique (saliva incubation) by comparing its discrimination power, in a morning bad breath inhibition study of antiseptics, to those of hydrogen sulphide (H2S) measurement devices and organoleptic ratings. METHODS: After a professional cleaning, 8 periodontally healthy students abstained from all means of mechanical plaque control for 5 experimental periods of 7 days, with intervening washout periods of at least 2 weeks. During each experimental period, the students rinsed only twice daily with different antiseptics. At day 7, morning breath was scored clinically (volatile sulphide compound [VSC] level and organoleptic ratings), and 1.5 ml of saliva was collected and divided between 3 glass tubes that were sealed and incubated (37 degrees C, anaerobic chamber). Immediately after collection and after 3 and 6 hours of incubation, the headspace air in one of the tubes was examined for VSC production and organoleptic measurements. RESULTS: The investigations of the incubated saliva correlated well with the 7-day intraoral VSC recordings and organoleptic ratings (P < or = 0.005). Moreover, evaluations showed a similar interproduct ranking for their efficacy in malodor control. The power analyses indicated a higher discrimination power for the saliva incubation test than for the intraoral registrations. CONCLUSIONS: The strong correlation between odor production of incubated saliva and clinical assessments suggests that the saliva incubation test may be used as an indirect method to measure oral malodor and can be employed to investigate the antimalodor effectiveness of oral hygiene products.

Adult↗
PubMed1997

Incubation temperature effects on physical characteristics of normal, dark, firm and dry, and halothane-carrier pork longissimus.

Pigs (n = 18) were selected to represent three different muscle conditions (six pigs per condition): normal: dark, firm, and dry; and halothane carrier. A 45-cm-long longissimus section was excised from each side of the carcass at 30 min postmortem and cut into six sections. Right side sections were assigned to the intermediate temperature incubation (23 degrees C), and left side sections were designated high temperature incubation (40 degrees C). Sections were randomly assigned to incubation times (0, 1, 2, 4, 6, or 8 h). The 0 h section from each incubation treatment was designated as a control and was placed directly into a 4 degree C cooler. Temperature and pH were evaluated on the control section and for each loin section a the end of the incubation time. Color (L*, a*, and b* values), percentage of purge loss, water-holding capacity, and drip loss were determined. Incubation treatment did not alter pH decline in dark, firm, and dry muscle; however, high temperature increased pH decline in normal and halothane carrier samples. Results suggest that there is a strong interaction between pH and temperature that affects pork quality attributes. High incubation temperature had a negative effect on most quality variables; however, muscle condition (normal or halothane carrier) had limited effects on muscle quality.

Animals↗
PubMed1996

Effect of ascorbic acid and cooling during egg incubation on hatchability, culling, mortality, and the body weights of broiler chickens.

Five experiments involving 3,100 settable eggs with living embryos of commercial broiler parent stock were conducted to determine the effect of ascorbic acid (AA) and cooling during egg incubation on embryonic weight, hatchability, percentage of cull chicks, embryonic mortality, and body weights of the hatched chicks. The treatments were carried out at 15, 15, 17, 11 and 19 d of incubation for Experiments 1, 2, 3, 4, and 5, respectively. The treatments for the experiments were as follows: 1) eggs injected with 0.1 mL of sterile saline solution; 2) eggs injected with saline containing 0.5, 1, 3, and 12 mg of AA per egg; and 3) control for Experiment 1; 1) eggs injected with 3 mg of AA per egg; 2) eggs cooled at 22 C for 24 h; and 3) control for Experiments 2 and 3; 1) eggs injected with 3 mg AA per egg; and 2) control for Experiments 4 and 5. Ascorbic acid at a dose of 3 mg during egg incubation improved hatchability and body weight, and decreased number of cull chicks, whereas a dose of 12 mg decreased embryo weight and hatchability and increased the number of cull chicks and embryonic mortality. Injection of AA hatchability improved when injected on Days 11 and 15 of incubation; effects diminished if AA was injected on Day 19 of incubation. Cooling eggs at 22 C for 24 h had no significant effect on any of the variables studied when compared with controls. Comparison of AA injection at a dose of 3 mg per egg and egg cooling to 22 C for 24 h at 15 to 17 d of incubation led to the conclusion that AA had two advantages over cooling. These advantages were 1) low embryonic death and 2) greater chick weight at hatch and on Days 15 and 17 of incubation.

Animals↗
PubMed1999

Evaluation of a cut-off value for sperm motility after different hours of incubation to select the suitable reproductive technology (IVF or ICSI).

BACKGROUND: The aim of this study was to enhance the predictability of conventional semen parameters for in-vitro fertilization outcome. The utility of late sperm motility in presence of a cumulus-oocyte complex after different hours of incubation was investigated to predict the outcome of IVF in borderline and normal ejaculates (at least 5 x 10(6) motile sperm). METHODS: The study was done on 52 infertile couples undergoing conventional in-vitro fertilization and embryo transfer. Sperm were prepared by the Percoll cushion centrifugation with swim-down. Cocultures were established by inseminating the cumulus-oocyte complexes with 100000 motile spermatozoa and incubating them for 48 hours. Another 100000 spermatozoa were incubated in culture medium for 48 hours. Sperm motility (WHO a+b) was determined at 0, 4, 24 and 48 hours of incubation. RESULTS: The fertilization rate was 65.5% (42.9-88.1). The conventional semen parameters did not correlate with the fertilization rate. Sperm motility measured after different hours of incubation was found to be significantly positively correlated with the fertilizing ability of sperm in vitro in Spearman's rank correlation test: motility after 0 h (p<0.02), after 4 h (p=0.0025). after 24 h (n.s.) and after 48 h (p=0.0071). Cut-off values for late sperm motility were determined and differences in fertilization rates were calculated for these cut-off values after different hours of incubation. A cut-off value of 20% progressive motile spermatozoa after 48 hours gave the best statistical power (fertilization rate 71.7 vs. 50.2%, p<0.001). Significant differences in the fertilization rates were also observed for a cut-off value of 35% after 24 hours of incubation (70.1 vs. 46.2%, p=0.001) and for a cut-off point of 60% after 4 hours (72.4 vs. 51.5%, p=0.001). CONCLUSIONS: The predictive power of sperm motility after 48 h for fertilization outcome provides support in the decision-making process within the assisted reproduction setting. If less than 20% of sperm are motile after 48 h micromanipulatory techniques should be considered.

Adult↗
PubMed2000

Mechanism of band 3 dimer dissociation during incubation of erythrocyte membranes at 37 degrees C.

The mechanism of dissociation of the stable dimer of band 3 was investigated during the incubation of isolated erythrocyte membranes or resealed ghosts at 37 degrees C. The kinetics of changes in the structural and functional integrity of the membrane domain of band 3 (MDB3) were measured and correlated with the change in the Stokes radius of band 3. MDB3 integrity was determined as follows: (1) by measuring the fluorescence emission spectrum of 4, 4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) bound covalently to MDB3; (2) by measuring the number of DIDS covalent binding sites present after incubation of unlabelled resealed ghosts; and (3) by measuring the anion transport V(max) by using the same resealed ghosts. Incubation of membranes at 37 degrees C caused the dissociation of band 3 dimers to monomers but only after a lag period lasting approx. 50 h. The observation of such a lag implies that dissociation involves a sequence of molecular events beginning with some type of initial process. We have discovered that this initial process involves a conformation change in MDB3. There was a shift in the fluorescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS binding capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 degrees C inhibited the conformational change in MDB3 and the dissociation of dimers. Furthermore, no conformational change in MDB3 was observed when erythrocytes were incubated at 37 degrees C. We suggest that MDB3 unfolding is the molecular event responsible for the subsequent dissociation of stable dimers of band 3 to monomers during the incubation of erythrocyte membranes at 37 degrees C. The monomers so generated are either not functional in anion exchange or they have an attenuated functionality. The absence of a conformational change for band 3 in erythrocytes might imply that haemolysis perturbs the membrane structure and somehow predisposes band 3 to the conformational change that occurs during incubation at 37 degrees C.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Antigen-binding activity of monoclonal antibodies after incubation with organic solvents.

Effects of four organic solvents--methanol, trifluoroethanol, dimethylsulfoxide, and dimethylformamide (DMF)--on the ferritin-binding activity of three monoclonal mouse antibodies of IgG2a and IgG1 subclasses were studied. The ferritin-binding constants of monoclonal antibodies G10 and F11 (the IgG2a subclass) were increased 2-6-fold after incubation with DMF and removal of the organic solvent by gel filtration. The maximum effect on the F11 antibodies was found in the presence of 5-13% DMF and on the G10 antibodies at 11-40% DMF. The effect remained after the removal of DMF from the incubation medium, and this suggests that the incubation with DMF resulted in irreversible conformational changes of the antibodies and in production of active conformers of the G10 and F11 antibodies. These conformations occurred within 15-60 min. The long-term stability and the fluorescence of the antibodies exposed to DMF suggest that the conformational changes were not global, but involved small and relatively independent structural elements of the antibodies, either of hypervariable CDR loops in variable domains or of the hinge region of the antibodies. The affinity of the C5 antibodies of the mouse IgG1 subclass was decreased after incubation with DMF. The activation was a solvent-specific effect because incubation of the G10 antibodies with methanol and dimethylsulfoxide decreased the affinity for the antigen, and incubation with trifluoroethanol virtually did not affect it. Relatively small changes in the antigen-binding activity of the antibodies were found even after the incubation with 5% organic solvent.

Animals↗