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PubMed2004

Incubation of cocaine craving after withdrawal: a review of preclinical data.

Using a rat model of drug craving and relapse, we recently found that cocaine seeking induced by re-exposure to drug-associated cues progressively increases over the first 2 months after withdrawal from cocaine self-administration, suggesting that drug craving incubates over time [Nature 412 (2001) 141]. Here, we summarize data from studies that further characterized this incubation phenomenon and briefly discuss its implications for drug addiction. The main findings of our ongoing research are: 1. Incubation of cocaine craving is long-lasting, but not permanent: cocaine seeking induced by exposure to cocaine cues remains elevated for up to 3 months of withdrawal, but decreases after 6 months. 2. Incubation of reward craving is not drug specific: sucrose seeking induced by re-exposure to the reward cues also increases after withdrawal, but for a time period that is shorter than that of cocaine. 3. Incubation of cocaine craving is not evident after acute re-exposure to cocaine itself: cocaine seeking induced by cocaine priming injections remains essentially unchanged over the first 6 months of withdrawal. 4. Incubation of cocaine craving after withdrawal is associated with increases in the levels of brain-derived neurotrophic factor (BDNF) in mesolimbic dopamine areas.

Amphetamine-Related Disorders↗

PubMed2005

Adjustments in IVF system for individual boars: value of additives and time of sperm-oocyte co-incubation.

In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5,943 COC's were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa from 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2mM), hyaluronic acid (HA; [0.5mg/mL]) and adenosine (10 microM), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5mg/ml) and adenosine (0, 10, 20 and 40 microM) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 min or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 12-15 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P<0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9+/-3.9% versus 62.7+/-3.9% and 1.5+/-3.2 versus 1.3+/-3.5 for 10 min or 6h, respectively), but reduced mono-spermy (P<0.001, 57.9+/-2.5% versus 70.0+/-2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF.

Animals↗

PubMed1978

Effects of ultraviolet irradiation and postirradiation incubation on heterogenous nuclear RNA size in murine cells.

We have analyzed the decrease in synthesis of individual size classes of heterogeneous nuclear RNA (hnRNA) in ultraviolet (UV)-irradiated Merwin plasmacytoma (MPC-11) cells at various times of postirradiation incubation. HnRNA from nonirradiated control cells is distributed over a wide range from approximately 60S to 5S, with 42S RNA carrying more label than any other size class. HnRNA from UV-irradiated cells shows a dose-dependent shift in size distribution toward lower molecular weight. The size distribution of hnRNA synthesized after prolonged times of postirradiation incubation is restored toward normal, i.e., synthesis of long RNA molecules increases relative to the synthesis of short ones. Analysis of the total number of hnRNA chains synthesized during a 20-min [(3)H]uridine pulse shows a considerable reduction in their number with increasing UV dose. Murine cell lines are excision-repair-deficient but capable of post replication repair inhibited by caffeine. HnRNA transcripts of cells incubated in its presence were studied. The caffeine, which has no effect on hnRNA size in control cells, inhibits to a considerable extent the restoration of full-length transcripts during postirradiation incubation. The lack of excision repair in MPC-11 was confirmed by the analysis of pyrimidine dimers in trichloracetic acid-insoluble and soluble fractions within 8 h of postirradiation incubation.The size of parental and daughter strand DNA in UV-irradiated cells was correlated with RNA transcript size. The parental DNA in these experiments does not change its size as a consequence of UV exposure and postirradiation incubation. In contrast, daughter DNA strands are short in UV-irradiated cells and they increase in size during postirradiation incubation to reach the size of parental strands after 8 h.

Animals↗

PubMed1994

Sperm motility, morphology, hyperactivation, and ionophore-induced acrosome reactions after overnight incubation with mycoplasmas.

OBJECTIVE: To evaluate the effect on the function on human sperm of overnight incubation with mycoplasma species. DESIGN: Prospective controlled in vitro study. SETTING: Community-based sperm bank and andrology laboratory. INTERVENTIONS: Percoll-separated sperm were incubated overnight with a low densities of Myco-plasma hominis and Ureaplasma urealyticum serotypes. MAIN OUTCOME MEASURES: Motility, midpiece and tail morphology, hyperactivation after overnight capacitation, and acrosome status after incubation with calcium ionophore. RESULTS: Overnight incubation with mycoplasma species resulted in small but highly statistically significant differences in motility, morphology, hyperactivation after overnight capacitation, and proportion acrosome reacted after incubation with calcium ionophore compared with controls. CONCLUSIONS: Incubation with mycoplasma species impairs sperm physiology. Identification and treatment of mycoplasma may augment a physiology-based infertility evaluation.

Acrosome↗

PubMed1998

Antisperm antibody treatment mode: levels of antisperm antibodies after incubation with TEST-yolk buffer and filtration using the SpermPrep II method.

OBJECTIVE: To assess whether incubation in TEST-yolk buffer (TYB) or human tubal fluid (HTF) could alter the sperm membrane characteristics and its relationship to antisperm antibodies (ASA) and/or antigen detachment from the sperm membrane and to evaluate the filtration of those specimens and possible recovery of ASA-free spermatozoa. DESIGN: A prospective clinical study. SETTING: Andrology Institute of Lexington, Lexington, Kentucky. PATIENT(S): Twenty patients undergoing infertility treatment. MAIN OUTCOME MEASURE(S): Recovery of spermatozoa with reduced levels or antisperm antibody-free sperm after treatment with TYB or HTF, followed by filtration using the SpermPrepII method (Sephadex based). RESULT(S): Assessment of ASA using the direct immunobead test showed no significant differences between specimens incubated for 2 hours in seminal plasma (fresh) or HTF with regard to levels of IgA and IgG. The percentage binding of anti-IgA and anti-IgG immunobeads was significantly reduced in specimens incubated for 2 hours in TYB compared with specimens incubated in seminal plasma or HTF. Furthermore, selection of spermatozoa using the SpermPrepII filtration method significantly reduced the percentage binding of anti-IgA and anti-IgG immunobeads compared with specimens incubated in HTF. CONCLUSION(S): The results suggest that TYB either altered the sperm membrane properties so that there was a decreased affinity at the antibody and/or antigen sites or that the egg yolk proteins were absorbing the antibodies and/or antigens complexes from the sperm membrane surface. Incubation of spermatozoa in TYB followed by filtration with the SpermPrepII method improved the recovery of ASA-free spermatozoa by selectively entrapping spermatozoa with ASA bound to its surface.

Antibodies, Anti-Idiotypic↗

PubMed1989

Enhanced progesterone and testosterone secretion and depressed estradiol secretion in vitro from small white follicle cells of incubating turkey hens.

This study was conducted to determine if lower steroid secretion by the small white follicles in incubating turkey hens contributes to lower circulating steroid concentrations during this time. Turkey hens were grouped as either laying or incubating. Serum samples and the ovarian small white follicles (SWF; 2-7 mm diameter) were collected from each hen. The SWF were pooled for each group and their cells were dispersed by trypsin digestion. Serum-luteinizing hormone (LH), progesterone (P), testosterone (T), and estradiol (E) concentrations were lower and serum prolactin concentration was higher during incubation than during egg laying. SWF cells from incubating hens secreted more P and T and less E in response to ovine luteinizing hormone (oLH) than did similar cell suspensions from laying hens. The incubating hens' SWF cells' capacity to secrete E but not their capacity to secrete P or T in vitro is consistent with the observed circulating levels. It is hypothesized that lower levels of circulating LH and/or higher levels of prolactin found in incubating hens may have a depressing effect on aromatase activity and/or an up-regulating effect on LH-induced P and T secretion by the SWF cells.

Animals↗

PubMed2002

Delayed incubation as an alternative method to sample storage for enumeration of E. coli and culturable bacteria in water.

The effects of sample storage on enumeration of Escherichia coli in marine bathing water and culturable bacteria in drinking water were evaluated. Results showed that overnight storage at 0-5 degrees C significantly reduced the counts of E. coli in bathing water (p = 0.0001) with a mean reduction of 25%. A similar effect of sample storage was observed for the enumeration of culturable bacteria in drinking water at 22 +/- 2 degrees C for 66 +/- 4 h (p = 0.0074; mean reduction = 25%) or at 36 +/- 2 degrees C for 44 +/- 4h (p = 0.0353; mean reduction = 6%). The use of a delayed incubation method, i.e. overnight storage at 0-5 degrees C of inoculated agar plates prior to incubation, did not significantly affect the counts of culturable bacteria when plates were incubated at 22 + 2 degrees C for 66 +/- 4 h, whereas it resulted in a significant increase of the bacterial numbers when plates were incubated at 36 +/- 2 degrees C for 44 +/- 4 h (p = 0.0002; mean increase = 32%). Based on these results, it is suggested to avoid the use of overnight or longer sample storage for the enumeration of E. coli in bathing water samples, as well as for the enumeration of culturable bacteria in drinking water. The delayed incubation method appears to be a reliable procedure for the enumeration of culturable bacteria and could represent a valid alternative to sample storage in order to overcome problems associated with the performance of bacteriological counts during weekends or statutory holidays. However, a multi-laboratory study is needed to evaluate the reproducibility of the delayed incubation method for the enumeration of culturable bacteria and its possible use for the enumeration of E. coli by membrane filtration.

Environmental Monitoring↗

PubMed1997

Sperm membrane functional integrity and response of frozen-thawed bovine spermatozoa during the hypoosmotic swelling test incubation at varying temperatures.

The objective of this study was to assess the sperm membrane integrity and permeability of frozen-thawed bovine spermatozoa, processed at varying temperatures during and after thawing, by exposing the spermatozoa to standardized hypoosmotic conditions. The hypoosmotic swelling (HOS) test was employed to measure changes in sperm membrane functional status and permeability. Frozen specimens (from 5 bulls) were thawed at 37h degrees C for 10 sec and transferred to a water bath at 37 (Aliquot 1), 21 (Aliquot 2) or 5 degrees C (Aliquot 3) to complete thawing (1 to 2 min). The specimens were maintained and processed at these temperatures for additional 5 to 10 min. Specimens were slowly diluted 1:1 (v/v) and washed with Ham's F-10 media containing 3% (w/v) BSA. The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.0 ml of a 100 mOsm/L HOS diluent. The following treatments were performed: 1) Aliquot 1 (control), specimens were incubated in HOS solutions at 37 degrees C for 5 min; 2) Aliquot 2, specimens were incubated in HOS solutions at 21 or 37 degrees C for 5 min; and 3) Aliquot 3, specimens were incubated in HOS solutions at 5 or 37 degrees C for 5 min. Samples were obtained from the sperm specimen-HOS diluent mixtures at 1 min intervals (during the 5 min incubation period), fixed and assessed for sperm swelling patterns. The sperm response to the HOS test for specimens processed at temperatures below 37 degrees C was higher when samples were incubated in HOS diluents at 37 degrees C. This finding indicates that the potential for sperm swelling (measurement of sperm membrane functional status) can be maintained when spermatozoa are processed at temperatures below 37 degrees C. The highest response to the HOS test was observed in spermatozoa processed at 21 degrees C and incubated in a HOS solution at 37 degrees C. The response to the HOS test was superior to the one observed in specimens maintained and processed at 37 degrees C throughout. Thawing of spermatozoa at 37 degrees C, followed by processing at 21 degrees C seems to reduce the negative effects associated with osmotic shock and results in the preservation of the sperm membrane functional status during the in vitro handling of frozen-thawed bovine spermatozoa.

Journal Article↗

PubMed1997

In vitro maturation of ovine oocytes in a portable incubator.

Immature ovine oocytes were collected from ovaries obtained from an abattoir and assigned to one of three treatment groups for in vitro maturation. For Treatment 1 (T1), oocytes were matured in a conventional incubator, in tissue culture wells in an atmosphere of 5% CO(2) and air. Maturation medium consisted of bicarbonate buffered Tissue Culture Medium 199 (TCM199) supplemented with fetal calf serum (FCS), follicle stimulating hormone (FSH), luteinizing hormone (LH), and penicillin/streptomycin (pen/strep). For Treatment 2 (T2), oocytes were matured in a portable incubator, in plastic tubes containing the same medium as T1. The medium was equilibrated with 5% CO(2) and overlayed with oil. For Treatment 3 (T3) oocytes were matured in the portable incubator without CO(2) equilibration, in tubes containing HEPES buffered TCM 199 supplemented as in T1. After 24 hours at 39 degrees C, the percentage of oocytes undergoing normal nuclear maturation was 72.55, 68.14 and 66.96% for T1, T2 and T3, respectively (P >0.05). In a second experiment oocytes were matured in the 3 treatments described, then fertilized in vitro using frozen-thawed ram sperm. Fertilization rates were 44.09, 58.62 and 55.69% for T1, T2 and T3, respectively. T1 and T2 were significantly different (P < 0.05). For Experiment 3, oocytes matured and fertilized as described were cultured in drops of Modified Brinster's Mouse Ova Culture (MBMOC) containing bovine oviductal cells. These were incubated at 39 degrees C in an atmosphere of 5% CO(2) and air for 7 days. T1, T2 and T3 resulted in 20.26, 16.94 and 24.43% development to morulae, and 4.01, 3.06 and 1.85% development to blastocysts, respectively (P >0.05). The results of these experiments indicate that maturation, fertilization, and developmental rates of ovine oocytes matured in the portable incubator are similar to those of oocytes matured in a conventional incubator. This technique shows promise for transportation of oocytes to laboratories where abattoirs are not in close proximity, and holds promise for transportation of oocytes from non-domestic animals collected in the field or remote locations, to facilities capable of utilizing and preserving the gametes.

Journal Article↗

PubMed2006

Kuru in the 21st century--an acquired human prion disease with very long incubation periods.

BACKGROUND: Kuru provides the principal experience of epidemic human prion disease. Its incidence has steadily fallen after the abrupt cessation of its route of transmission (endocannibalism) in Papua New Guinea in the 1950s. The onset of variant Creutzfeldt-Jakob disease (vCJD), and the unknown prevalence of infection after the extensive dietary exposure to bovine spongiform encephalopathy (BSE) prions in the UK, has led to renewed interest in kuru. We investigated possible incubation periods, pathogenesis, and genetic susceptibility factors in kuru patients in Papua New Guinea. METHODS: We strengthened active kuru surveillance in 1996 with an expanded field team to investigate all suspected patients. Detailed histories of residence and exposure to mortuary feasts were obtained together with serial neurological examination, if possible. FINDINGS: We identified 11 patients with kuru from July, 1996, to June, 2004, all living in the South Fore. All patients were born before the cessation of cannibalism in the late 1950s. The minimum estimated incubation periods ranged from 34 to 41 years. However, likely incubation periods in men ranged from 39 to 56 years and could have been up to 7 years longer. PRNP analysis showed that most patients with kuru were heterozygous at polymorphic codon 129, a genotype associated with extended incubation periods and resistance to prion disease. INTERPRETATION: Incubation periods of infection with human prions can exceed 50 years. In human infection with BSE prions, species-barrier effects, which are characteristic of cross-species transmission, would be expected to further increase the mean and range of incubation periods, compared with recycling of prions within species. These data should inform attempts to model variant CJD epidemiology.

Aged↗

PubMed1984

Prolonged incubation improves the micro-scale in-vitro test for drug sensitivity of Plasmodium falciparum.

In the standard micro-scale in-vitro test for detecting resistance of Plasmodium falciparum to chloroquine, isolates are incubated for 24-28 h in medium that does not contain serum. The effect of prolonging incubation to 48 h and of adding human AB serum to the culture medium of P falciparum from Irian Jaya, Indonesia was studied. Addition of serum was not associated with any advantages or disadvantages. Production of schizonts was increased when incubation was prolonged. This facilitated reading of the malaria smears and interpretation of the results. The percentage of successful tests (greater than or equal to 20 schizonts/200 asexual parasites in control wells) increased from 31% at 28 h incubation to 94% at 48 h. Among the 7 (44%) isolates which were chloroquine-resistant in vivo, only 1 met the criteria for a positive test at 28 h incubation, but at 48 h all 7 did. To achieve optimum results from the micro-scale in-vitro test in Jayapura, Irian Jaya, isolates should be incubated for 48 h.

Adolescent↗

PubMed1994

Release of lipid from the equine placenta during in vitro incubation.

An in vitro incubation technique was used to examine release of lipids from the equine placenta. Placental tissue was obtained at term (n = 5, term = 320-365 days) and earlier in gestation (n = 8, mean = 266 days). Term placentae were incubated at two temperatures, 4 degrees C (control) and 37 degrees C for 2 h. Pre-term placentae were incubated at 37 degrees C with two different concentrations of fatty acid in the medium. Tissues and media were analysed for their lipid concentrations. Term and pre-term placentae released free fatty acid (FFA) and phospholipid into the incubation medium during incubation at 37 degrees C. Long chain polyunsaturated fatty acids derived from the essential fatty acids were released into the media. The fatty acid profiles of the lipids released during incubation more closely resembled those of fetal plasma than maternal plasma lipids as measured in previous studies. These data are consistent with the view that the equine placenta is a source of both FFA and phospholipid for the fetus and that the placenta may provide long chain polyunsaturated fatty acids for the fetal foal.

Animals↗

PubMed2001

In vitro co-incubation of pig islet cells with xenogeneic human blood mononuclear cells causes loss of insulin release during perifusion: involvement of non-T-cell- and T-cell-mediated mechanisms.

Because the different steps of the human cellular immune rejection of pig islets are still poorly understood, our previous work concerned the intensity and mechanisms of the proliferation of human peripheral blood mononuclear cells (PBMC) to adult pig islet cells (PIC). As lymphocyte proliferation is not indicative of alteration of PIC, the present in vitro study evaluated cell-mediated immune effectors possibly involved in impairment of adult PIC. A test was thus developed, based on perifusion analysis of the alteration of insulin release from PIC incubated with different human cells. Compared to PIC incubation alone or with autologous pig splenocytes, seven-day co-incubation with whole human peripheral blood mononuclear cells (PBMC) (n = 18) led to almost complete abolition of basal and stimulated insulin releases (p < 0.0001). This effect could not be reversed by extensive sequential washes before perifusion of PIC, and the number of PIC was decreased by 78% after seven-day co-incubation with PBMC. PBMC are a complex mixture of cells involved in different xenogeneic mechanisms, and two components of this PIC impairment were then detected separately. First, the effect of PBMC against PIC was decreased (p < 0.0001) after removal of either MHC class II+ or CD14+ cells from PBMC. On the contrary, decreasing effect (p < 0.001) on insulin secretion was observed when only plastic-adherent or CD14+ cells were co-incubated with PIC. Additionally, alteration of insulin release from PIC cultured with PBMC or plastic-adherent cells was abolished dose-dependently (p < 0.0001 and p < 0.04, respectively) by gadolinium chloride (which inhibits macrophages), but not modified by cyclosporin A or mycophenolate mofetil which did not alter insulin release from PIC but blocked the proliferation of PBMC against PIC. A second mechanism was also detected, since co-incubation of PIC with purified human T cells remixed with antigen-presenting cells led to a decrease (p < 0.0001) of insulin release. This model based on the alteration of dynamic basal and stimulated insulin secretion provides detailed account of in vitro human cell-mediated impairment of PIC. It shows that the xenogeneic effect of whole mononuclear cells was strong and rapid. A crucial role was played by MHC class II+, CD14+, and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: 1) early direct effect, potentially involved in vivo in primary nonfunction of islets aggressed by monocytes/macrophages; and 2) the presentation of PIC xenoantigens leading to impairment by T lymphocytes, which may be involved in in vivo specific cellular rejection.

Animals↗

PubMed2002

Influence of CO(2) incubation on quinolone activity against Streptococcus pneumoniae and Haemophilus influenzae.

Quinolone activity can be influenced by the pH change that occurs during CO(2) incubation when testing capnophilic organisms such as Streptococcus pneumoniae and Haemophilus influenzae. This study compares the activity of ciprofloxacin, clinafloxacin, enrofloxacin, fleroxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, pefloxacin, sparfloxacin, trovafloxacin, and the glycopeptide vancomycin, under ambient air and supplemental CO(2) incubation conditions. Etest (AB BIODISK, Solna, Sweden) was used to determine the MIC values of 30 S. pneumoniae strains including S. pneumoniae ATCC 49619 and 6030; and 29 H. influenzae strains including H. influenzae ATCC 49247 and 49766, incubated with and without 5% CO(2.) Reference broth microdilution and agar dilution tests were performed under similar conditions. Results determined that MIC values of all quinolones agreed (> or = 95%) within +/- one log(2) dilution for the three test methods tested under identical incubation conditions. Quinolone MICs for S. pneumoniae were minimally influenced by CO(2,) while MIC values for H. influenzae ranged from 0.5 to two log(2) dilutions higher when incubated in the CO(2) environment. The differences in quinolone activity against H. influenzae may be due to a combination of pH effects and enhanced growth in the CO(2) test conditions. Quality control and interpretative criteria, especially for H. influenzae and quinolones published by the National Committee for Clinical Laboratory Standards, may not be fully applicable to results obtained under CO(2) incubation. This may generate potential therapeutic interpretive dilemmas in the susceptibility testing of H. influenzae, where clinical isolates require CO(2) to sustain acceptable growth.

4-Quinolones↗

PubMed2001

Serum levels of HCV RNA and core protein before and after incubation at 37 degrees C for 24 h.

The kinetics of HCV during interferon (IFN) therapy have recently been described and the estimated virion half-life is an average of 2.7 h, suggesting that HCV infection is highly dynamic. The aim of this study was to evaluate serum levels of HCV-RNA and HCV core protein (HCV-Ag) before and after incubation at 37 degrees C for 24 h. We also evaluated the viral kinetics during IFN treatment by determining their serum levels at 0, 24 and 48 h, and day 8 after the start of treatment. The decay slope was calculated as the logarithm of the ratio of HCV-RNA levels at 0 and 24 h of incubation: log(virus load) 24 h-log(virus load) 0 h and the estimated half-life was also calculated. The decay slope was -1.66+/-0.75 (-4.12 to -0.18) (mean+/-S.D. (range)) and the estimated virion half-life was 6.2+/-6.9 h (1.8-39.3). The HCV-RNA level was rapidly decreased to 6.8+/-13.1% of the initial load after incubation independently of the serotype. In contrast, the HCV-Ag level after incubation for 24 h was 98.7+/-12.2% of the initial level. The synthesized naked HCV-RNA (equivalent to 10(7) copy/ml) was not detected after 1-min incubation. These data suggested that HCV virions are very unstable and collapsed rapidly and that HCV-RNA, existing outside of virions, is immediately degraded in serum, whereas HCV-Ag remains stable. IFN treatment caused a rapid decrease in the levels of both HCV-RNA and HCV-Ag. The HCV-RNA decay slope was -1.95+/-0.96 (range: -3.48 to -0.50) and was similar to that seen in the incubation study. Our result suggested the significance of measuring HCV-Ag during clinical management independently of HCV-RNA, especially because of its high stability.

Journal Article↗

PubMed2005

Kinetics of formation and functional properties of conjugates prepared by dry-state incubation of beta-lactoglobulin/acacia gum electrostatic complexes.

The formation of conjugates between beta-lactoglobulin and acacia gum based on electrostatic complexes formed at pH 4.2 was investigated upon dry-state incubation for up to 14 days at 60 degrees C and 79% relative humidity (RH). By means of SEC-HPLC and RP-HPLC, it was shown that the beta-lactoglobulin incubated alone was able to form polymers with molecular masses higher than 200 kDa until 50% of the initial monomeric protein disappeared after 14 days. In the presence of acacia gum at initial protein to polysaccharide weight mixing ratios of 2:1 and 1:2, only 35% of the initial beta-lactoglobulin monomers disappeared after 14 days. Using RP-HPLC, an apparent reaction order of 2 was found for the disappearance of monomeric beta-lactoglobulin both in the presence or absence of acacia gum. However, the reaction rate was faster in the absence of acacia gum. SDS-PAGE electrophoresis with silver staining confirmed the formation of beta-lactoglobulin/acacia gum conjugates. The solubility curves of the incubated beta-lactoglobulin showed a minimum around pH 4-5. By contrast, the minimum of solubility of the beta-lactoglobulin/acacia gum incubated mixtures shifted to lower pH values compared to initial mixtures. The conjugates exhibited higher foam capacity than the incubated protein as well as lower equilibrium air/water surface tension. Conjugation at ratio 1:2 led to increased interfacial viscosity (300 mN s m(-1) at 0.01 Hz) compared to beta-lactoglobulin alone (100 mN s m(-1) at 0.01 Hz), but similar interfacial elasticity (30-40 mN m(-1)). The foam capacity of the conjugates was significantly higher than that of the incubated beta-lactoglobulin as well as foam expansion and drainage time, especially at pH 5.3, i.e., higher than the pH of formation of the conjugates.

Chemical Phenomena↗

PubMed1992

Availability of caesium isotopes in vegetation estimated from incubation and extraction experiments.

A rapid and inexpensive incubation and extraction technique for estimating the available low molecular weight and potentially bioavailable fraction of Cs isotopes in vegetation has been developed. Samples of contaminated vegetation (grass, herbage and fungi) and litter were exposed to rumen liquid from sheep, by in vitro incubation or by placing nylon bags in the rumen of sheep (in sacco). The results were compared with laboratory extractions using rumen buffer, NH4OAc, CsCl and de-ionized water. The release of low molecular weight 137Cs species after in vitro incubation with rumen liquid for 4-8 h was 75-85% for grass, herbage and fungi, 30% for lichen and 10% for litter. The reproducibility of the method was 5% for digestible matrices and 10-20% for litter where the fraction of Cs isotopes released was small. Extractions with NH4OAc and CsCl released 75 and 80% of the Cs isotopes in grass, respectively. Significantly lower extraction yields were obtained with de-ionized water and buffer. During in sacco incubation, 90-100% of the Cs isotopes in vegetation was released within 1 h; however, potentially available low molecular weight forms and Cs-bearing colloidal material could not be differentiated. The results obtained by incubation with rumen liquid and by extraction with NH4OAc or CsCl were in good agreement with published data from a 3 week feeding trial. For practical reasons extraction with NH4OAc or CsCl rather than incubation with rumen liquid is recommended for estimating the fraction of available low molecular weight Cs species.(ABSTRACT TRUNCATED AT 250 WORDS)

Accidents↗

PubMed2005

The temperature dependence of porphyrin production in Propionibacterium acnes after incubation with 5-aminolevulinic acid (ALA) and its methyl ester (m-ALA).

Topical PDT treatment of the common skin disease acne vulgaris is now in clinical use. Propionibacterium acnes (P. acnes) is known to play an important role in acne. 5-Aminolevulinic acid (ALA) supplementation leads to an enhanced porphyrin production in the bacteria. Subsequent illumination with light of the proper wavelengths can reduce the number of bacteria and this might at least partly explain the PDT effect on acne. We have assessed the effects of temperature on P. acnes washed cell suspensions incubated for 4 h with ALA or ALA methyl ester (m-ALA). The effect on porphyrin production of both the cell suspension incubation temperature as well as the initial growth temperature of the cultivated cells prior to harvesting and use in suspension experiments was investigated. The bacterial porphyrin content was estimated from fluorescence emission spectra. It was found that incubation with ALA or m-ALA at a temperature 42 degrees C resulted in an approx. 100% and 33% increase in the total amount of PDT-relevant porphyrins produced as compared to incubation at 37 degrees C. These results support increasing the skin temperature during incubation with ALA or m-ALA in the clinic. The initial growth temperature, prior to the incubation, had no apparent effect on the ALA or m-ALA induced porphyrins. Activation energy studies indicate slightly higher temperature dependence in the case of ALA produced porphyrins as compared to m-ALA produced porphyrins (77 and 65 kJ mol(-1), respectively).

Aminolevulinic Acid↗