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PubMed1995

Species variation in the bioactivation of tacrine by hepatic microsomes.

1. The metabolite profile of tacrine (1,2,3,4-tetrahydro-9-amino acridine) was similar in hepatic microsomes from man, rat, dog, rabbit, mouse and hamster. Major metabolites were 1-, 2-, 4- and 7-OH tacrine. Only quantitative differences in metabolite profile were evident between species. 2. Bioactivation to protein-reactive metabolite(s) was seen in microsomes from all species. 3. 7-Methyl tacrine was found to undergo significantly less bioactivation than either 7-OH tacrine or tacrine itself. 4. In the presence of hepatic microsomes and thiol-containing agents protein-reactive metabolite formation was significantly reduced. With mercaptoethanol present a stable thioether adduct was generated from both tacrine and 7-OH tacrine. 5. Analysis of the thioether adduct by mass spectrometry yielded a molecular ion of m/z 290 consistent with the presence of a covalent adduct of 7-OH tacrine complexed in a 1:1 molar ratio with mercaptoethanol. 6. We have therefore provided further evidence for a two-step mechanism in the bioactivation of tacrine involving an initial 7-hydroxylation followed by a postulated 2-electron oxidation to yield a reactive quinone methide. This mechanism of bioactivation appears to be identical in human and animal hepatic microsomes.

Amino Acids↗

PubMed1995

Role of hepatic metabolism in the bioactivation and detoxication of amodiaquine.

1. The hepatic metabolism of the antimalarial drug amodiaquine was investigated in order to gain further insight into the postulated metabolic causation of the hepatotoxicity, which restricts the use of the drug. After intraportal (i.p.) administration (54 mumol/kg) to the anaesthetized rat, the drug was excreted in bile (23 +/- 3% dose over 5 h; mean +/- SD, n = 6) primarily as thioether conjugates. 2. After i.p. administration, 20% of the dose was excreted into urine over 24 h as parent compound and products of N-dealkylation and oxidative deamination. Desethylamodiaquine accumulated in liver, but was not a substrate for bioactivation as measured by biliary elimination of a glutathione adduct. 3. Prior administration of ketoconazole, an inhibitor of P450, reduced biliary excretion by 50% and effected a corresponding decrease in the amount of drug irreversibly bound to liver proteins. This indicated a role for P450 in the bioactivation of amodiaquine to a reactive metabolite that conjugates with glutathione and protein. 4. De-ethylation and irreversible binding were observed in vitro using male rat liver microsomes, and were again inhibited by ketoconazole. However, no such binding was observed with human (six individuals) hepatic microsomes despite extensive turnover of amodiaquine to desethylamodiaquine. 5. Amodiaquine quinoneimine underwent rapid reduction in the presence of either human or rat liver microsomes. Therefore in vitro studies may underestimate the bioactivation of amodiaquine in vivo. These data indicate that the extent of protein adduct formation in the liver will depend on the relative rates of oxidation of amodiaquine and reduction of its quinoneimine. This in turn may be a predisposing factor in the idiosyncratic hepatotoxicity associated with amodiaquine. 6. Substitution of a fluorine for the phenolic hydroxyl group in amodiaquine blocked bioactivation of the drug in vivo. Insertion of an N-hydroxyethyl function enabled partial clearance of amodiaquine and its deshydroxyfluoro analogue via O-glucuronidation and altered the balance between phase I oxidation and direct phase II conjugation of amodiaquine.

Amodiaquine↗

PubMed2000

Bioactive milk peptides: a prospectus.

Bioactive peptides have been identified within the amino acid sequences of native milk proteins. Hydrolytic reactions, such as those catalyzed by digestive enzymes, result in their release. These peptides directly influence numerous biological processes evoking behavioral, gastrointestinal, hormonal, immunological, neurological, and nutritional responses. The specific bioreactions associated with each physiological class have been well characterized. Herein, we review the scientific literature and attempt to stimulate consideration of the continued use of bioactive peptides and their expanded development as a commercial product. Several applications have already evolved. For example, phosphopeptides derived from casein fractions are currently used as both dietary and pharmaceutical supplements. Potentially, the addition of bioactive peptides to food products could improve consumer safety as a result of their antimicrobial properties. Lastly, bioactive peptides may function as health care products, providing therapeutic value for either treatment of infection or prevention of disease.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2000

Bioactivation of tegafur to 5-fluorouracil is catalyzed by cytochrome P-450 2A6 in human liver microsomes in vitro.

Tegafur is a prodrug of 5-fluorouracil (5-FU) consisting of a new class of oral chemotherapeutic agents, tegafur/uracil and S-1, which are classified as dihydropyrimidine dehydrogenase inhibitory fluoropyrimidines. It is bioactivated to 5-FU via 5'-hydroxylation mediated by cytochrome P-450 (CYP). However, which isoform(s) of CYP is responsible for the bioactivation process of tegafur remains unclear. The purpose of the present study was to identify the human CYP isoform(s) involved in the metabolic activation of tegafur using human liver microsomes and cDNA-expressed human CYPs. The formation of 5-FU from tegafur in human liver microsomes showed biphase kinetics with Km and Vmax values for the high-affinity component of 0.43 +/- 0.05 mM and 4.02 +/- 1.70 nmol/mg/min (mean +/- SD, n = 4), respectively. In the correlation study using a panel of 10 human liver microsomes, the formation of 5-FU from tegafur showed a significant correlation (r = 0.98; P < 0.001) with coumarin 7-hydroxylation, a marker activity of CYP2A6. In addition, a specific substrate of CYP2A6 and anti-CYP2A6 antibody inhibited the formation of 5-FU by 90% in human liver microsomes. Moreover, cDNA-expressed CYP2A6 showed the highest activity for the formation of 5-FU among 10 cDNA-expressed CYPs, with a Km value similar to that found for the high-affinity component in human liver microsomes. These findings clearly suggest that CYP2A6 is a principal enzyme responsible for the bioactivation process of tegafur in human liver microsomes. However, to what extent the bioactivation of tegafur by CYP2A6 accounts for the formation of 5-FU in vivo remains unclear, because the formation of 5-FU from tegafur is also catalyzed by the soluble fraction of a 100,000 x g supernatant and also derived from spontaneous degradation of tegafur.

Antimetabolites, Antineoplastic↗

PubMed1999

Treatment of dental root fracture by medium energy CO2 laser and DP-bioactive glass paste. Part. II: Compositional, structure, and phase changes of DP-bioglass paste after exposed to CO2 laser.

Fractured teeth are difficult to treat effectively. Currently, such as root fractures are usually treated by full-coverage restoration, root amputation, or tooth extraction. If the fusion of tooth fracture by laser were possible, it could offer a different therapy to repair fracture teeth. We tried to use a developed DP-bioactive glass paste to fuse or bridge the tooth crack line by a medium energy continuous-wave CO2 laser. The present report was focused on the phase transformation and rescrystallization of DP-bioactive paste during expose to the CO2 laser. The materials will examine by means of XRD, FTIR, DTA/TGA, and SEM. From the study, we could expect that the temperature increase due to laser irradiation must be over than 900 degrees C and the DP-bioactive glass paste could be melted in a short period of time after irradiation. In the study, we successfully developed a DP-bioactive glass paste which could form a melting glass within minutes after exposed to a medium energy density continuous-wave CO2 laser. The paste will be used to bridge the enamel or dentine surface crack by the continuous-wave CO2 laser in the near future.

Dental Cements↗

PubMed2002

[Bioactive compounds from marine sponges and cell culture of marine sponges].

Presented a survey of bioactive compounds discovered from marine sponges in the recent five years, including the classes, distribution and their potential pharmaceutical uses. In particular, the compounds with antitumor, antivirus and antibacteria activity were discussed with their originating marine sponge species. Whereas the "Supply Problems" were identified to hinder the clinical tests and commercial applications of most of the sponge bioactive compounds. In vitro cell culture of marine sponges is one of the most promising approaches to solve this problem. The state-of-the art of marine sponge cell culture and the challenging areas were discussed. A brief summary of the R&D status was also given on the bioactive compounds from marine sponges in Chinese oceans. It is crucial to invest more efforts on studying marine sponges and their bioactive compounds in our country in order to develop new marine drugs of independent intellectual property.

Animals↗

PubMed2002

[Use of bioactive glass ceramics in the treatment of tibial plateau fractures].

PURPOSE OF THE STUDY: The surgical treatment of proximal tibia fractures requires a reduction and fixation of the split condyle, elevation of the depressed interfragment of articular surface. The golden standard is the application of autogenous bone grafts for filling in the defect in the cancellous bone of the tibial condyle. Bioactive glass-ceramics material may be successfully used for the filling. Gass-ceramics BAS-O is biocompatible and bioactive and has also suitable mechanical properties and therefore it was used in combination with autogenous bone marrow in order to speed up osteogenesis. MATERIAL: The study covered 12 patients, 4 men and 8 women, with a fracture of the proximal tibia classified according to AO as type B3.1, B3.2 and C3.2. The average follow-up period was 5.5 years (range, 2 to 8 years). METHOD: After the assessment of the type of fracture and revision of soft tissues the depressed interfragment of articular surface was elevated and the defect was completely filled with BAS-O granules in combination with 10 ml of autogenous bone marrow. The broken condyle was fixed by screws or buttress plate. During the first year the process of healing was checked by radiographs at 3-month intervals. All 12 patients were invited for a follow-up examination where the radiograph of the proximal tibia was assessed in two projections and the clinical condition was evaluated using Rasmussen criteria. RESULTS: According to the Rasmussen criteria 12 patients achieved the value of 21-29 points with the average of 26.4 of 30 possible points. The volume of the BAS-O filling was 7.1-44.3 ccm and did not change throughout the whole period of the follow-up. The depression of an interfragment of articular surface prior to operation was 3-17 mm, average 6.3 mm. The residual depression after the operation was 0-4 mm, average 1.7 mm. No symptoms of loosening, extrusion or surrounding of the BAS-O material by fibrous tissue was found. The integration with the surrounding bone occurred during the first 3 months. DISCUSSION: The bioactive coral hydroxyapatite has been successfully used to fill similar defects by a number of authors. Among the benefits of BAS-O granular system is an easy handling and a fast integration with the bone tissue of the recipient due to the development of a hydroxyapatite layer on its surface after the contact with tissue fluid. Autologous bone marrow was added to the system in order to initiate osteogenesis and a faster healing of the defect. The mentioned material is suitable also from the mechanical viewpoint as neither deformation nor resorption was found in the followed-up patients. We reduced the surgery duration and morbidity and improved the patients comfort. CONCLUSIONS: Part of the surgical management of the fracture of the tibial plateau with a depressed segment of subchondral bone is its elevation. For filling in of the traumatic defect in the cancellous bone of the proximal tibia the bioactive, glass-ceramics, granular material BAS-O may be successfully used. Mechanical and chemical properties (compression strength, non-resorption) ensure its stable position in the filling as well as a constant volume. Osteointegration was accelerated by adding 10 ml of autologous bone marrow. The use of the glass-ceramics material reduces the surgery duration, improves the patient's comfort and decreases the probability of incidence of complications.

Adult↗

PubMed2002

[Studies on the stabilities of bioactive selenocompounds in selenium-enriched garlic and onion].

It is reported that selenium (Se) incorporation into garlic increases the bioactivities of garlic. Hence, the chemical changes of selenocompounds during processing and storage will influence the bioactive effectiveness of Se-enriched garlic. The principal selenocompounds in Se-enriched garlic are water-soluble, and several comparative experiments are thus conducted to examine the stabilities of the selenocompounds in water extracts of Se-enriched garlic and Se-enriched onion. The results show that preparing the garlic powder by freeze-drying technique can maintain the chemical properties of the selenium compounds in Se-enriched garlic. Se-methyl-selenocysteine is unstable in water extract of Se-enriched garlic when the extract is prepared and stored at room temperature. Specific alliinase inhibitor hydroxylamine (NHOH.HCl) effectively prevents the loss of Se-methyl-selenocysteine, which suggests that the decomposition of Se-methyl-selenocysteine may be catalyzed by alliinase. Se-methyl-selenocysteine is one of the main bioactive selenocompounds in Se-enriched garlic. The procedures of processing and storage should be carefully chosen to prevent the loss of selenocompounds and the decrease of the bioactivity of Se-enriched garlic. Se-enriched onion also contains alliinase and Se-methyl-selenocysteine, but its Se-methyl-selenocysteine is proved to be stable in the same water extract as that of Se-enriched garlic. The stability differences of Se-methyl-selenocysteine in Se-enriched garlic and onion, the mechanism of selenocompound decomposition and the bio-activities of decomposed compounds in Se-enriched garlic need to be further studied.

Carbon-Sulfur Lyases↗

PubMedDate not supplied

Modulation of immunoradiometric and bioactive follicle stimulating hormone secretion and clearance in young and elderly men during treatment with tamoxifen or flutamide.

The secretion and clearance of immunoactive and bioactive follicle-stimulating hormone (FSH) in healthy young men (N = 10) and elderly men (N = 7) during blockade of endogenous sex steroid hormones with tamoxifen, an antiestrogen, and flutamide, an antiandrogen, was investigated. To this end, subjects underwent blood sampling basally every 10 minutes for 24 hours, and then received 2 consecutive intravenous pulses of synthetic gonadotropin releasing hormone (GnRH; 10 micrograms and 100 micrograms) every 2 hours. This paradigm was repeated on two subsequent visits, in which subjects received either flutamide HCl, a specific nonsteroidal competitive antagonist of the androgen receptor (750 mg daily for 3 days), or tamoxifen, a selective antagonist of the estrogen receptor (20 mg daily for 9 days). Serum immunoactive FSH concentrations were measured in each sample by immunoradiometric assay (IRMA). Serum bioactive FSH concentrations were determined by an in vitro bioassay (rat granulosa cell aromatase system) on 24-hour serum pools. Deconvolution analysis was used to analyze both the FSH IRMA 24-hour time series and FSH release after GnRH. Comparisons between young and elderly men of the basal state showed significantly increased 24-hour mean serum immunoactive and bioactive FSH concentrations and significantly decreased free testosterone concentrations in elderly men. By deconvolution analysis, elderly men had a significant decrease in FSH secretory burst duration, and an increase in FSH half-life and FSH secretory burst amplitude compared with younger men. In response to sex steroid receptor blockade in young men, there was a significant increase in mean serum bioactive FSH concentrations during antiandrogen treatment, but not during antiestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMedDate not supplied

Utilization of autogenous bone, bioactive glasses, and calcium phosphate cement in surgical mandibular bone defects in Cebus apella monkeys.

PURPOSE: The purpose of the present study was to evaluate the histologic results of bone cavities that were surgically created in the mandibles of Cebus apella monkeys and filled with autogenous bone, PerioGlas, FillerBone, or Bone Source. MATERIALS AND METHODS: Surgical cavities 5 mm in diameter were prepared through both mandibular cortices in the mandibular angle region. The cavities were randomly filled, and the animals were divided into groups according to the material employed: Group 1 cavities were filled with autogenous corticocancellous bone; group 2 cavities were filled with calcium phosphate cement (BoneSource); and group 3 and group 4 cavities were filled with bioactive glass (FillerBone and PerioGlas, respectively). After 180 days the animals were sacrificed, and specimens were prepared following routine laboratory procedures for hematoxylin/eosin staining and histologic evaluation. RESULTS: The histologic analysis showed that autogenous bone allowed total repair of the bone defects; bioactive glasses (FillerBone and PerioGlas) allowed total repair of the defects with intimate contact of the remaining granules and newly formed bone; and the cavities filled with calcium phosphate cement (BoneSource) were generally filled by connective fibrous tissue, and the material was almost totally resorbed. DISCUSSION: The autogenous bone, FillerBone, and PerioGlas provided results similar to those in the current literature, showing that autogenous bone is the best choice for filling critical-size defects. Synthetic implanted materials demonstrated biocompatibility, but the bio-glasses demonstrated osteoconductive activity that did not occur with calcium phosphate (Bone-Source). CONCLUSION: According to the methodology used in this study, it can be concluded that the utilization of autogenous bone and bioactive glasses permitted the repair of surgically created critical-size defects by newly formed bone; the synthetic implanted materials demonstrated biocompatibility, and the bioactive glasses demonstrated osteoconductive activity. The PerioGlas was mostly resorbed and replaced by bone and the remaining granules were in close contact with bone; the FillerBone showed many granules in contact with the newly formed bone; BoneSource did not permit repair of the critical-size defects, and the defects were generally filled by connective fibrous tissue.

Animals↗

PubMed2003

[Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471].

AIM: To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system. METHODS: The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry. RESULTS: The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha. CONCLUSION: The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.

Animals↗

PubMed2004

Identification of an N-methyl-4-phenylpyridinium-like metabolite of the antidiarrheal agent loperamide in human liver microsomes: underlying reason(s) for the lack of neurotoxicity despite the bioactivation event.

In contrast with the Parkinson's-like effects associated with the mitochondrial neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroleptic agent haloperidol, there exist no reports on adverse central nervous system (CNS) effects with the structurally related N-substituted-4-arylpiperidin-4-ol derivative and antidiarrheal agent loperamide. Although this difference can be attributed to loperamide's P-glycoprotein substrate properties that prevent it from accessing the brain, an alternative possibility is that loperamide metabolism in humans is different from that of MPTP and haloperidol and does not involve bioactivation to a neurotoxic pyridinium species. In the current study, loperamide bioactivation was examined with particular focus on identification of pyridinium metabolites. A NADPH-dependent disappearance of loperamide was observed in both rat and human liver microsomes (human t(1/2) = 13 min; rat t(1/2) = 22 min). Loperamide metabolism was similar in human and rat and involved N-dealkylation to N-desmethylloperamide (M3) as the principal metabolic fate. Other routes of loperamide biotransformation included N- and C-hydroxylation to the loperamide-N-oxide (M4) and carbinolamide (M2) metabolites, respectively. Furthermore, the formation of an additional metabolite (M5) was also discernible in human and rat liver microsomes. The structure of M5 was assigned to the pyridinium species (LPP(+)) based on comparison of the liquid chromatography/tandem mass spectrometry characteristics to the pyridinium obtained from loperamide via a chemical reaction. Loperamide metabolism in human microsomes was sensitive to ketoconazole and bupropion treatment, suggesting P4503A4 and -2B6 involvement. Recombinant P4503A4 catalyzed all of the loperamide biotransformation pathways in human liver microsomes, whereas P4502B6 was only responsible for N-dealkylation and N-oxidation routes. The wide safety margin of loperamide (compared with MPTP and haloperidol) despite metabolism to a potentially neurotoxic pyridinium species likely stems from a combination of factors that include a therapeutic regimen normally restricted to a few days and the fact that loperamide and perhaps LPP(+) are P-glycoprotein substrates and are denied entry into the CNS. The differences in safety profile of haloperidol and loperamide despite a common bioactivation event supports the notion that not all compounds undergoing bioactivation in vitro will necessarily elicit a toxicological response in vivo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

[Chemical study on bioactive natural products of novel structural types].

Bioactive natural products of novel structural types play a highly significant role in the drug discovery and development process. Of the 1,031 new chemical entities covering all diseases in the years 1981-2002, 48% were natural product mimics, 5% were the direct utility of natural products as sources of novel structures. Up to now, the main challenges are more efficient and rapid processes for discovery and isolation of bioactive natural products of novel structural types. Therefore, new strategies and ideas for chemical study on bioactive natural products of novel structural types should be applied. In order to discover a large number of bioactive natural products of novel structural types from naturally occurring sources and elucidate the intrinsic value of natural products as leads to new structures with different activities, new, more efficient and rapid processes for study on natural products will be established by use of modern chromatographies, techniques on line identification of natural products and screening methods based on molecular targets.

Biological Products↗

PubMed2005

[Bioactive peptides derived from milk proteins].

Milk proteins are known for having a wide range of nutritional, functional and biological properties that make them important ingredients in functional or health promoting foods. These properties are partly attributed to bioactive peptides coded in the different milk proteins. Bioactive peptides are inactive within the protein sequence but may be released by the action of native proteolitic enzymes from milk, enzymes from lactic acid bacteria or from exogenous sources or may be produced during gastrointestinal digestion or processing of foods. Peptides derived from caseins and whey proteins were shown to present several bioactive properties such as opioid, antihypertensive, antimicrobial, immunodulatory, mineral carrier and antithrombotic. This overview presents a perspective of the importance of dairy proteins in the production of bioactive peptides and their biological activities, as well as the main analytical tecniques that have been used for the isolation and identification of these peptides.

Milk Proteins↗

PubMed1991

Comparison between the synthetic cholecystokinin analogues caerulein and Thr28NlE31CCK25-33(CCK9) with regards to plasma bioactivity, degradation rate and stimulation of pancreatic exocrine function.

A new synthetic analogue of cholecystokinin, Thr28Nle31CCK25-33(CCK9) is compared with caerulein with regards to plasma bioactivity, degradation rate, side effects, and stimulation of pancreatic secretion. 24 healthy male volunteers were intubated with a double lumen Lagerlöf-tube. 30 min after correct positioning of the tube subjects received a continuous intravenous infusion of synthetic secretin (1 U/kg) together with either ceruletide (61.5 pM/kg) or CCK9 (30 pM/kg) both for 45 min. 30 pM/kg of CCK9 have been shown by others to cause maximal enzyme secretion (1). 61.5 pM/kg (= 100 ng) of caerulein are probably supramaximal but used by many centers for direct pancreatic function tests. Plasma CCK was measured by bioassay which compares amylase release of isolated rat pancreatic acini stimulated by plasma extracts with known standards of CCK8. Lipase, amylase, trypsin, chymotrypsin, and bicarbonate were measured in 15 min fractions after the onset of CCK infusion. Both drugs caused a similar stimulation of pancreatic enzyme secretion during infusion of the respective analogue which declined after termination. After the onset of CCK9 infusion plasma bioactivity reached a plateau around 20 pM at 15 min. Values declined after 30 min. After termination of infusion bioactivity rapidly declined within 3 min but still remained slightly elevated after further 15 min as compared to basal values (3.9 vs 0.6 pM). Plasma kinetics of caerulein were quite similar. However, despite a dose which was only twice as high as compared to CCK9, plasma bioactivity was six times higher with plateau values of about 120 pM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2006

[Application of NF-kappaB reporter and Dual-Luciferase assays in the measure of bioactivity of interleukin-1beta and interleukin-1 receptor antagonist].

OBJECTIVE: To develop a reporter gene system based on transient transfections with a NF-kappaB responsive reporter gene to detect the bioactivity of IL-1beta and IL-1 receptor antagonist. METHODS: NF-kappaB reporter and Dual-Luciferase assays were applied to measure the bioactivity of IL-1beta and IL-1 receptor antagonist in mouse EL4 cells (some subclones of EL4 cells expressed high level of IL-1 receptor on cell surface). pNF-kappaB-luc and pRL-TK, used as an internal control, were co-transfected into EL4 cells and then the IL-1beta was added. RESULTS: The results indicated that IL-1beta was able to induce the expression of this luciferase, which could be blocked by IL-1 receptor antagonist. The optimal dose of IL-1beta was 5 microg/L in Dual-Luciferase assay, whose bioactivity can be effectively inhibited by IL-1ra at 50 microg/L. CONCLUSION: We have established a new method to detect the bioactivity of IL-1beta and IL-1 receptor antagonist, which can give repeatable results.

Animals↗

PubMed1991

Single proline substitutions in predicted alpha-helices of murine granulocyte-macrophage colony-stimulating factor result in a loss in bioactivity and altered glycosylation.

Contributions of alpha-helices to biological activity in murine granulocyte-macrophage colony-stimulating factor were analyzed using site-directed mutagenesis and protein expression in COS-1 cells. A series of single proline substitutions were made for residues within the four predicted alpha-helices as a means of disrupting local helical secondary structure. Mutations in three of the four helices resulted in marked reductions in bioactivity. Five mutants E21P, L56P, E60P, L63P, and L107P showed 10(2)-10(4)-fold reduction in bioactivity as well as hyperglycosylation. The same Pro substitutions made on non-N-glycosylated molecules had a similar loss in bioactivity implying that a Pro-induced structural change and not hyperglycosylation was responsible for the major decrease in bioactivity. Additional amino acid substitutions at these residues which conserved charge or hydrophobicity, or replaced the original residue with an Ala, verified that conformational changes in the protein structure were specifically due to steric constraints imposed by the Pro residue rather than loss of important side chain functions.

Amino Acid Sequence↗

PubMed1990

The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity.

The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.

Animals↗