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PubMed1994

Incubation temperature affects the behavior of adult leopard geckos (Eublepharis macularius).

The leopard gecko has temperature-dependent sex determination (TSD); females are predominantly produced when incubated at 26 degrees C (100%), 30 degrees C (70%), and 34 degrees C (95%), whereas males are predominantly produced at 32.5 degrees C (75%). Exogenous estradiol can override the effect of temperature on sex determination. To compare temperature-determined females with hormone-determined females, eggs from the male-biased temperature were treated with estradiol benzoate during incubation. As adults, animals from a male-biased incubation temperature were more likely to exhibit aggression than animals from female-biased incubation temperatures. Furthermore, females from a male-biased incubation temperature tended to be less attractive than females from female-biased temperatures. Hormone-determined females were both attractive and aggressive. This suggests that incubation temperature is an important development determinant of adult aggressiveness and attractiveness. The 26 degrees C animals ovariectomized on the day of hatch exhibited more frequent aggression and were unreceptive to males, indicating that postnatal ovarian hormones also play a role in adult sociosexual behaviors. The parallel between incubation temperature and intrauterine position in laboratory mammals is discussed.

Aggression↗
PubMed1981

Cortisol protection of the granulocyte response to isoproterenol during an in vitro influenza virus incubation.

Isolated human polymorphonuclear leukocytes (PMNs) release beta glucuronidase (BG) lysosomal enzyme during incubation with complement-activated zymosan particles. In asthma, isoproterenol (ISO) and histamine (HIS) inhibition of PMN lysosomal enzyme release is impaired while the prostaglandin E1 (PGE1) response is normal. During a respiratory infection provoking asthma, the PMN response to ISO is further impaired. The PMN response to ISO, HIS, and PGE1 is similarly impaired following an in vitro incubation with a live influenza bivalent (A + B) vaccine. In the following study, granulocytes were isolated from normal (n = 10) and asthma patients (n = 29). The isolated cells were incubated with the bivalent influenza vaccine (1 PMN:1 egg infective dose50) for 30 min. Following this in vitro virus incubation, granulocytes from normal and asthma patients had an imparied response to ISO, HIS, and PGE1. Granulocytes from asthma patients having a history of wheezing with respiratory illnesses did not have a greater impairment in the agonist response following virus incubation than normals or allergic asthma patients. However, in asthma patients (n = 6) receiving prednisone (10 to 20 mg/day), the in vitro vaccine incubation did not impair the PMN ISO response. A similar protective effect was found if granulocytes were incubated with hydrocortisone (1,4 X 10(-4) M) prior to exposure to the influenza vaccine.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1987

Distinct prion proteins in short and long scrapie incubation period mice.

The Prn-i gene, controlling scrapie incubation period, is linked to or congruent with the murine prion protein (PrP) gene, Prn-p. In prototypic mouse strains with long (l/Ln) and short (NZW) incubation periods, Prn-p transcription is initiated at similar multiple sites. The predicted NZW and l/Ln PrP proteins differ at codons 108 and 189. Codon 189, highly conserved in mammals, lies within a polymorphic BstEll site that is retained in 17 mouse strains known to have short or intermediate incubation times, but is absent in l/Ln and two other inbred mice with long incubation times. Codon 108 in mice with short or intermediate incubation times encodes Leu; in mice with long incubation times it encodes Phe. The correlation of PrP sequence with length of scrapie incubation period suggests, but does not formally prove, congruency between Prn-p and Prn-i.

Amino Acid Sequence↗
PubMed2005

In vitro penetration of fresh and vitrified swine oocytes by homologous spermatozoa using different incubation systems.

The present study consisted of two experiments. In the first one, ejaculates from four boars were used to compare in vitro penetration (IVP) rates of fresh and vitrified swine oocytes by homologous spermatozoa in four treatments: fresh oocytes in conventional incubation (CO2 incubator) (FC), vitrified oocytes in conventional incubation (VC), fresh oocytes in submarine (bag) incubation (FS) and vitrified oocytes in submarine incubation (VS). The IVP rates for FC, VC, FS and VS were 46.5, 44.3, 36.9 and 33.1%, respectively. Analysis through Chi-square tests identified no differences in IVP rates between FC and VC and between FS and VS (P > 0.05), but IVP rate for FC was greater (P < 0.05) than those for both FS and VS. Besides IVP rate for VC did not differ (P > 0.05) from those for FC and FS, but it was greater than that for VS (P < 0.05). Logistic regression analysis identified differential effects of treatments dependant on individual boars. The second experiment evaluated the influence of semen storage period on the semen quality of the two boars associated with greater IVP rate in the first experiment. Semen quality was estimated by IVP rate using the VC treatment and by the following methods: sperm motility, sperm morphology, hypoosmotic swelling test (HOST) and thermal stress test (TST). According to analysis using Chi-square tests, IVP rate did not differ (P > 0.05), for the first boar, between 0 (100.0%) and 24 h of semen storage (98.1%) nor after 48 and 72 h (66.0 and 59.3%, respectively), but IVP rates were greater during the 0-24 h period compared with the 48-72 h period (P < 0.05). For the second boar, IVP rate at 0 h (50.6%) was greater (P < 0.05) than at 24, 48 and 72 h of semen storage (34.3, 28.3 and 24.0%, respectively), with no further differences observed after 24 h (P > 0.05). Logistic regression analysis identified that the effect of storage on IVP rate was influenced by the effect of individual boars. No differences in semen quality during the storage period were identified by conventional methods of semen evaluation, for either boar (P > 0.05) using analysis of variance with repeated measures. These results indicate that IVP test can be used to estimate boar fertility, even when vitrified oocytes are used (if using conventional CO2 incubators) or using an alternative submarine incubation system (if using fresh oocytes). The IVP test was the only method of semen evaluation that identified the reduction in semen quality up to 72 h of storage.

Analysis of Variance↗
PubMed2006

The impact of incubating the throat culture for 72 h on the diagnosis of group A beta-hemolytic streptococci.

OBJECTIVE: The accurate detection and treatment of group A beta-hemolytic streptococcal pharyngitis is important to prevent the potential sequels. Throat culture remains the gold standard for the diagnosis of streptococcal pharyngitis. Many authors recommended 48 h incubation time for the isolation of group A Streptococcus (GAS). In this study the diagnostic efficacy of prolonging the incubation time of throat cultures to 72 h for isolation of GAS was researched. METHODS: Throat swab specimens were inoculated to 5% sheep blood agar plate and incubated at 37 degrees C, and evaluated at the 24th hour. GAS negative plates were incubated again for another 24h and read at the 48th hour. Negative plates at 48 h were incubated again, read at the 72nd hour, and positive results of all measurements were evaluated. RESULTS: A total of 367 cultures were examined. At the 24th hour evaluation 34 (9.3%) cultures were positive for GAS. Evaluation of 333 cultures that were found to be negative in the first 24th hour evaluation revealed 44 (13.6%) positive cultures at 48th hour evaluation. Out of 289 cultures that were negative during the 48th hour evaluation, an additional 13 (4.7%) cultures were found to be positive during the 72nd hour evaluation. The agreement between 24th-48th and 24th and 72nd were both moderate (kappa 0.54 and 0. 46, respectively), and between 48th and 72nd hour was very good (kappa: 0.90). CONCLUSION: Throat cultures that are negative for GAS at 24h should be incubated for second day. We conclude that, to confirm the throat cultures GAS negative, results of throat cultures need to be incubated for 72 h, since 72nd hour evaluations revealed additional 5% positive results.

Adolescent↗
PubMed2004

Effects of incubation on solubility and mobility of trace metals in two contaminated soils.

Much research has focused on changes in solubility and mobility of trace metals in soils under incubation. In this experiment, changes in solubility and mobility of trace metals (Pb, Cu and As) and Fe in two contaminated soils from Tampa, Florida and Montreal, Canada were examined. Soils of 30 g were packed in columns and were incubated for 3-80 days under water-flooding incubation. Following incubation, metal concentrations in pore water (water soluble) and in 0.01 M CaCl2 leachates (exchangeable+water soluble) were determined. While both soils were contaminated with Pb (1600-2500 mg kg(-1)), Tampa soil was also contaminated with As (230 mg kg(-1)). Contrast to the low pH (3.8) of Tampa soil, Montreal soil had an alkaline pH of 7.7 and high Ca of 1.6%. Concentrations of Fe(II) increased with incubation time in the Tampa soil mainly due to reductive Fe dissolution, but decreased in the Montreal soil possibly due to formation of FeCO3. The inverse relationship between concentrations of Pb and Fe(II) in pore water coupled with the fact that Fe(II) concentrations were much greater than those of Pb in pore water may suggest the importance of Fe(II) in controlling Pb solubility in soils. However, changes in concentrations of Fe(II), Pb, Cu and As in pore water with incubation time were similar to those in leachate, i.e. water soluble metals were positively related to exchangeable metals in the two contaminated soils. This research suggests the importance of Fe in controlling metal solubility and mobility in soils under water-flooded incubation.

Arsenic↗
PubMed2005

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions.

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode's complete medium (TCM; a capacitating medium), Ca2+-free TCM (TCM-Ca2+), or Ca2+ and NaHCO3-free TCM (Tyrode's basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3h period of incubation at 38 degrees C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern (r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa (r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.

Animals↗
PubMed2007

Estimates of short- and long-term incubation periods of Plasmodium vivax malaria in the Republic of Korea.

With the current epidemic of vivax malaria closely associated with the demilitarised zone along the border between North and South Korea, it has been suggested that the incubation period tends, in part, to be prolonged. Based on the detailed travel history of cases from 2000 to 2003 who reside in non-malarious areas, statistical estimates of the incubation periods were obtained. The data suggest that cases fall into two categories with short- and long-term incubation periods, respectively. Of 416 cases with available information, 72 and 79 successfully met our criteria for inferring the durations of short- and long-term incubation periods. The mean short- and long-term incubation periods were estimated to be 26.6 days (95% CI 21.0-32.2) and 48.2 weeks (95% CI 46.8-49.5), respectively. The maximum likelihood method was used to fit gamma and normal distributions to the short- and long-term incubation periods, assisting prediction of the frequency distribution of the overall incubation period, which exhibited a bimodal pattern. We postulate that the observed distribution reflects adaptation of the parasite to the seasonal population dynamics of the vector, Anopheles sinensis, ensuring continued transmission of vivax malaria in this temperate zone.

Adolescent↗
PubMed2005

Effects of gonadal sex and incubation temperature on the ontogeny of gonadal steroid concentrations and secondary sex structures in leopard geckos, Eublepharis macularius.

Incubation temperature during embryonic development determines gonadal sex in the leopard gecko (Eublepharis macularius). Incubation temperature and gonadal sex jointly influence the display of sexual and agonistic behavior in adult leopard geckos. These differences in adult behavior are organized prior to sexual maturity, and it is plausible that post-natal hormones influence neural and behavioral differentiation. Here we assessed incubation temperature and sex effects on sex steroid levels in leopard geckos at 2, 10, and 25 weeks of age and monitored the development of male secondary sex structures. Males had significantly higher androgen concentrations at all time points, whereas females had significantly higher 17beta-estradiol (E2) concentrations only at 10 and 25 weeks. Within males, age but not incubation temperature affected steroid levels and morphological development. Male androgen levels increased modestly by 10 and dramatically by 25 weeks of age, whereas E2 levels remained unchanged over this period. Most males had signs of hemipenes at 10 weeks of age, and all males had hemipenes and open preanal pores by 25 weeks of age. In females, age and incubation temperature affected E2 and dihydrotestosterone (DHT) but not T concentrations. Controlling for age, females from 34 degrees C have higher DHT and lower E2 levels than females from 30 degrees C. Further, E2 concentrations increased significantly from 2 to 10 weeks, after which E2 levels remained steady. Together, these results indicate that sexually dimorphic levels of steroids play a major role in the development of leopard gecko behavior and morphology. Furthermore, these data suggest that the organizational effects of incubation temperature on adult female phenotype could be, in part, mediated by incubation temperature effects on steroid hormone levels during juvenile development.

Age Factors↗
PubMed2002

Effect of incubating human sperm at room temperature on capacitation-related events.

OBJECTIVE: To determine the effect of human sperm incubation at room temperature (20 degrees C) upon capacitation-related events. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Semen samples were obtained from normozoospermic volunteers. Human follicular fluid (hFF) was collected from women undergoing assisted reproductive treatment. INTERVENTION(S): Spermatozoa were incubated for up to 18 hours at 20 degrees C and/or 37 degrees C. MAIN OUTCOME MEASURE(S): Protein tyrosine phosphorylation patterns, development of hyperactivated motility, and induction of acrosome reaction (AR) in response to hFF. RESULT(S): Spermatozoa incubated for 18 hours at 20 degrees C showed an array of tyrosine phosphorylated proteins similar to noncapacitated cells. After incubation at 20 degrees C, the percentage of spermatozoa displaying hyperactivated motility and undergoing acrosomal loss in response to hFF was significantly lower when compared with cells kept the same time at 37 degrees C. Conversely, spermatozoa incubated overnight at 37 degrees C could respond to hFF, either at 37 degrees C or 20 degrees C. When preincubation at 20 degrees C was followed by sperm exposure to 37 degrees C, capacitation-related events could be activated. In capacitated cells (16 hours at 37 degrees C), 2-hour incubation at 20 degrees C led to a significant decrease in acrosome reaction inducibility, suggesting sperm decapacitation. CONCLUSION(S): Human sperm incubation at room temperature does not allow capacitation, although it does not affect hFF-induced acrosome reaction in capacitated cells. The blocking effect is overcome when spermatozoa are exposed to 37 degrees C.

Acrosome Reaction↗
PubMed1987

Incubation periods of cancer: old and new.

Following a review of the early efforts in occupational studies and animal experiments that were useful to elucidate concepts of latency and incubation periods in cancer, the paper presents the problem of definitions. In addition to semantic issues, conceptual models have influenced the development of appropriate empirical research in this field. The problems of measurement facing studies of incubation periods include; the assessment of time and nature of exposure, the definition of disease onset and the variation of sources of data used in the estimation of these periods. The different approaches that have been used to measure the incubation periods of cancer include; the direct approach, a study of changes of relative risks with time, calculation of time sequence between an outbreak of disease and the introduction of a suspected exposure, population based incidence studies, analysis of anecdotal information and studies of case clusters using different methods of establishing case linkages. There are well defined practical applications for the study of incubation periods of cancer. These are primarily in the area of etiological investigation, or may have practical relevance in community and patient management in past and future risk assessment. In its final part the paper raises issues that are researcheable in this subject. Does the incubation period of a cancer vary with the different etiologies in a multifactorial model? What is the effect of the dose of carcinogen on the length of the incubation period? Does the incubation period vary with age?

Animals↗
PubMed2002

Comparison of the effects of using standard and simple portable CO2 incubators on the in vitro developmental competence of bovine embryos reconstituted by somatic cell nuclear transfer.

In an attempt to determine the cultural factors that would improve cloning efficiency, we compared the effects of two incubation systems-a simple portable system and a standard CO2 incubator-on the production of bovine embryos by electrofusion of quiescent fetal fibroblast nuclei to enucleated oocytes matured in vitro. While the temperature (38.5 degrees C) and CO2 concentration (5%) were similar in both systems, the portable incubator operated in a vacuum of 300 mmHg and at an O2 level of 8-10%, which is lower than the standard. Although there were no significant differences between the two systems in terms of in vitro oocyte maturation (MII stage), fusion rates, and the number of cells in Day 7 blastocysts, significantly higher proportions of nuclear-transferred oocytes cleaved (P < 0.05) and developed to the blastocyst stage (P < 0.01) in the portable incubator (70.5 +/- 0.6 and 36.1 +/- 1.4%, respectively) than in the standard incubator (64.1 +/- 3.2 and 23.5 +/- 1.4%, respectively). Following the transfer of six blastocysts from the portable incubator group to three recipients, survival rates on Days 60, 90, and 120 were 100, 66.7 and 33.3%, respectively. This relatively high early embryonic loss may be associated with multiple pregnancy complications or other abnormalities of placentation frequently observed in cloned embryos. Further studies using this portable incubator system are needed to determine the optimum levels of O2, CO2, and air pressure.

Animals↗
PubMed2000

Optimization of a precision-cut trout liver tissue slice assay as a screen for vitellogenin induction: comparison of slice incubation techniques.

An in vitro male rainbow trout liver slice assay has been developed for long-term incubation of precision-cut slices for the detection of vitellogenin (VTG) protein induction in response to xenobiotic chemicals. The assay was optimized to allow 72 h of incubation of slices to maximize detection of VTG, while maintaining slice viability. Two methods of incubation frequently used with rat liver slices were compared: (1) slices were submerged in media (11 degrees C) and cultured in 12-well plates (PL) with continuous shaking; or (2) slices were floated onto titanium screens, placed into glass vials, and held under dynamic organ culture (DOC) conditions (11 degrees C). Slices (200 µm) in modified L-15 media were exposed to 1.0 µM 17beta-estradiol (E2) or diethylstilbestrol (DES). Protein from media and slice was sampled for Western blot analysis, using a polyclonal antibody to detect appearance of VTG protein. Maximum VTG was seen at 72 h, with detectable protein at 24 and 48 h in slices and media following PL incubation. In contrast, slices incubated in DOC showed little detectable VTG above background levels after 72 h. This difference was not attributable to protein loss to vial or plate surfaces. Standard viability assays did not reveal any differences between slices incubated in PL or DOC. However, histopathological examination revealed earlier and more severe vacuolization in slices incubated in DOC. Significantly more E2 uptake and conversion to water-soluble metabolites was noted in PL, compared with DOC, as well as more production of VTG in response to DES and E2, correlated with less histologic change. The in vitro assay described allows tissue-level assessment of estrogenicity in aquatic organisms, and will be useful for assessing not only comparative species receptor binding and transactivation, but also the role of tissue-specific activation factors in the estrogenic response of fish.

Journal Article↗
PubMed2002

The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.

Animals↗
PubMed2004

Changes of fatty acid composition in incubated rat pancreatic islets.

OBJECTIVE: The hypothesis that changes in fatty acId composition of pancreatic islets occur during incubation was investigated. METHODS: The content and composition of fatty acIds (FA) from rat pancreatic islets and culture medium after incubation for 1 and 3 hours in the absence or in the presence of 5.6, 8.3, or 16.7 mM glucose were determined by HPLC analysis. RESULTS: The FA content of pancreatic islets was reduced after 1 hour incubation in the absence of glucose. However, the total FA content was restored by incubating in the presence of 5.6 mM glucose and exceeded by incubating in the presence of 8.3 mM or 16.7 mM glucose. Saturated FA contributed a substantially greater proportion of the total FA increase in comparison to unsaturated FA, being palmitic and stearic acIds the most important. The total lipId content of pancreatic islets was not increased if the period of incubation in the presence of glucose was extended to 3 hours. A substantial amount of FA was found in the medium after 1 hour incubation in the absence of glucose: 141 ng per 80 islets for saturated and 75 ng per 80 islets for unsaturated. The release of FA from islets is increased in the presence of glucose. CONCLUSION: The release of FA from islets is a novel finding and may be related to modulation of B-cell function.

Animals↗
PubMed2003

The composition of the incubation medium influences the sensitivity of mitochondrial permeability transition to cyclosporin A.

The aim of this work was to study permeability transition, and the influence of the composition of the incubation medium, on the inhibitory action of cyclosporin A. It was found that cyclosporin inhibited the opening of a nonspecific pore, as induced by the uncoupler carbonyl cyanide m-chlorophenylhydrazone, provided K+ was present in the incubation medium, but failed to do so if mitochondria are incubated in sucrose or Na+-based medium. It was also found that the sensitivity of mitochondria to the uncoupler depended on the incubation mixture, being more sensitive when sucrose was the osmotic support. Matrix Ca2+ release, large amplitude swelling, and drop in transmembrane electric gradient revealed permeability transition. The titration of membrane thiol groups shows them to be increased in mitochondria incubated in sucrose medium, in comparison with the values found in mitochondria incubated in KCl or NaCl medium. Our proposal is that the incubation in sucrose medium propitiated a conformational change of membrane proteins in such a way that cyclosporin was unable to bind to its target site.

Calcium↗
PubMed2003

Gamma III-crystallin is the primary target of glycation in the bovine lens incubated under physiological conditions.

Several mechanisms have been proposed for the way in which glucose and its metabolites cause cataract, retinopathy and other complications of diabetes, the most convincing being glycation. Glycation, the reaction of sugars with free amino groups of proteins, is one of a variety of non-enzymic post-translational modifications. The aim of the present study was to identify some of the most reactive proteins in the lens when incubated under physiological conditions. Fresh intact bovine lenses were incubated with [14C]glucose in a conventional tissue-culture medium with added antibiotics. After 3 and 6 days of incubation, the water-soluble proteins were separated by size-exclusion chromatography. Glycated proteins from the water-soluble fractions were separated by using a sugar affinity column (Affi-Gel 601). Then the radioactive fractions were identified on SDS/polyacrylamide gels. In addition, the whole bovine lenses were incubated with 10 mM fructose and glucose for 3 and 6 days. The glycated proteins from the water-soluble fractions in parallel with the radioactive fractions were separated by affinity chromatography, and were identified further by amino-acid sequencing. A progressive uptake of radioactive label showed that the majority of proteins incorporating both glucose and fructose were water-soluble fractions. Chromatography and SDS/polyacrylamide gel results showed that alpha- and gamma-crystallin and some proteins of a mean molecular mass of 36-37 kDa incorporated sugars early during incubation. After 6 days of incubation, more crystallins were glycated compared with 3 days, in particular beta-crystallin. Affinity-chromatography results indicated that proteins with subunit masses of 36 kDa and 20 kDa were possibly radiolabelled at an early stage. The purified glycated proteins following incubation with both glucose and fructose, which corresponded to 20 kDa and 36 kDa bands on SDS/polyacrylamide gels, were sequenced by Edman degradation. N-terminal sequences of both 20 kDa bands were Gly-Lys-Ile-Thr, characteristic of gamma-crystallins, but the N-termini of both 36 kDa bands were blocked. Further sequencing after digestion of 36 kDa bands with trypsin and running on HPLC revealed that the glucose sample gave the peptide sequences as Gly-Glu-Tyr-Pro-Asp-Tyr-Gln-Gln and Tyr-Glu-Leu-Pro-Asn-Tyr-Arg, which match with bovine gammaIIIb-crystallin. The peptide sequence Tyr-Glu-Leu-Pro-Asn-Tyr-Arg is only present in the published sequence of bovine gammaIIIb-crystallin and not in any other type of gamma-crystallin. The fructose sample gave the peptide sequences Ile-Thr-Phe-Tyr-Glu-Asp-Arg, Arg-Gly-Asp-Tyr-Pro-Asp-Tyr-Gln-Gln-Trp, Gln-Tyr-Leu-Leu-Arg and Val-Val-Asp-Leu-Tyr, which all matched with bovine gammaIIIa-crystallin. The sequence Val-Val-Asp-Leu-Tyr only appears in the sequence of bovine gammaIIIa-crystallin. gammaIII-Crystallin is the most susceptible lens protein to glycation. The primary target of glucose is gammaIIIb-crystallin, whereas that of fructose is gammaIIIa-crystallin. The early glycation of gammaIII-crystallin by glucose and fructose could result in structural alterations, leading to aggregation of crystallin and eventually cataract formation.

Amino Acid Sequence↗
PubMed1991

Heparin-binding serum protein(s) is required for the protection of sialyltransferase released during the incubation of rat jejunal slices.

Incubation of rat jejunal slices in Krebs-Ringer bicarbonate buffer (KRB) required the presence of heat-inactivated horse serum (HHS) in order to show time-dependent release of sialyltransferase into the medium. Sialyltransferase activity could not be detected in the medium when KRB alone or KRB supplemented with either albumin or glycerol was used in the incubations. The viability of the jejunal slices for up to 4 h of incubation was determined by studying the incorporation of glucosamine and leucine into acid-insoluble proteins. Supplementation of KRB with HHS had no beneficial effect on the rate of incorporation of leucine and glucosamine into proteins. KRB medium obtained after different periods of incubation contained higher trypsin-like activity than KRB medium containing HHS. Various antiproteases present as supplements to KRB resulted in the release of sialyltransferase activity from the jejunal slices. Among these antiproteases, alpha 1-proteinase inhibitor (alpha 1-PI) was the most effective. Also, HHS added to KRB immediately following incubation resulted in partial restoration of sialyltransferase activity in the medium, suggesting the presence of anti-proteolytic factors in HHS. The addition of increasing concentrations of heparin to incubations containing HHS caused a decrease in the medium sialyltransferase activity. The heparin-binding fraction (HBF) from HHS, when added to incubations, was able to protect the sialyltransferase released into medium. However, HHS depleted of its heparin-binding fraction by heparin-agarose affinity chromatography was unable to protect the sialyltransferase. HBF was separated into high- and low-molecular-mass fractions (fractions A and B respectively) by gel-filtration chromatography. The capacity to protect the released sialyltransferase was contained in fraction B. Fraction A contained multiple bands on SDS/PAGE and did not protect the enzyme. Fraction B contained a major protein band on the gel which corresponded to the migration of a similar band in human alpha 1-PI. HBF as well as fraction B isolated from HHS showed anti-trypsin-like activity. The results presented indicate that HHS contains a heparin-binding protein(s) similar to human alpha 1-PI which plays a role in the protection of sialyltransferase released from jejunal slices.

Animals↗