Retinoic acid receptors (RARs) consist of six domain structures. The C-terminal region (D/E/F-domains) is involved in ligand binding, dimerization, and ligand-dependent transactivation. Structural information about RARs is required for understanding its complex function. A photoreactive retinoid denoted as ADAM-3, which was designed as the result of comparison of two fluorescent retinoids (DAM-3 and DAM-15), was synthesized and used for photoaffinity labeling of recombinant protein MBP-RARα/E. The photoaffinity-labeled site was determined by an endoprotease combination method which utilizes four endoproteinases in a two-phase digestion procedure. Two major labeled fragments were detected in each digestion, and the results of two-phase digestion allowed identification of the labeled residues as being located within residues 492-510 and 585-594, which correspond to 288-306 and 381-390 in human RARα, respectively.
Two typical systems of lipid peroxidation in egg yolk phosphatidylcholine (egg PC) liposomes were compared : an enzymic system involving superoxide (O-2) generated by xanthine (X), xanthine oxidase (XO) and Fe3+-chelates (Fe3+-ADP and Fe3+-EDTA), and a non-enzymic system involing ascorbic acid (AsA) and Fe2+. These two systems exhibited a different pH-dependence : the rate in the enzymic system was maximal at pH 8-8.5, wheareas that in the non-enzymic system was high below pH 7.4 and low above pH 7.6. The rates of lipid peroxidation differed with the membrane charge, and this charge-dependent phenomenon differed in the two peroxidation systems : in the Fe3+-chelates/X-XO-system, the rate was slow in neutrally charged egg PC liposomes and rapid in egg PC liposomes containing negatively charged dicetylphosphate (DCP) or positively charged stearylamine (SA), whereas in the Fe2+/AsA-system, the rate was rapid in neutral egg PC liposomes but no lipid peroxidation occurred in egg PC liposomes charged with DCP or SA. The decomposition rate of the hydroperoxide of PC (PC-OOH) incorporated into dimyristoyl-phosphatidylcholine (DMPC) liposomes differed depending on the membrane charge in the two systems and this charge-dependence of the rates correlated well with that of the initiation rate of lipid peroxidation dependent on membrane charge. In the Fe2+/AsA-system, lipid peroxidation depended on the endogenous presence of PC-OOH, and the amounts of PC-OOH required for initiation of the reaction differed depending on the membrane charge. However, in the Fe3+-chelates/X-XO-system, lipid peroxidation occurred very slowly in the absence of PC-OOH, but rapidly in its presence.
Recombinant human tumor necrosis factor (TNF) was digested with endopeptidases under mild conditions. Incubation of the TNF (155-amino-acid TNF) with trypsin, Staphylococcus aureus V-8 protease or chymotrypsin initially released small peptides derived from the amino (N)-terminal region of TNF, but did not release peptides from the carboxyl (C)-terminal region. The TNF was resistant to carboxypeptidases A and Y under a non-denaturing condition, but in the presence of urea or sodium dodecyl sulfate the C-terminal amino acid was released quantitatively by these peptidases. These results indicate that the N-terminal region of the TNF molecule is accessible to protease, while the C-terminal region is not susceptible to degradation.When the TNF was incubated with seven kinds of endopeptidases, its activity rapidly disappeared. At an early stage of the degradation, one active fragment was detected among the fragments produced with trypsin or pronase P, but no active fragments were detected on the degradation with the other peptidases. The active fragment was a fragment lacking the four N-terminal amino acid residues of the TNF. These results suggest that TNF is initially degraded at the N-terminal region by an endopeptidase and loses its activity as the degradation proceeds.
The mechanism of the antioxidant action of thiopalmitic acid (SH-Pal) was examined in an in vitro system measuring ferric (Fe(III))-nitrilotriacetate (NTA)- and Fe(III)-NTA/ascorbic acid (AsA)-induced lipid peroxidation of rat liver phospholipid liposomes and microsomes. The extent of lipid peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARS). SH-Pal and glutathione (GSH) scarcely stimulated the Fe(III)-NTA-induced lipid peroxidation in contrast with the mode of action, being similar to those produced by reducing-agent antioxidants such as cysteine and AsA. SH-Pal reduced iron similar to the action produced by AsA and cysteine, but not that of GSH under the same conditions. Also, the reduction of iron by SH-Pal did not exhibit a pH dependency. Similarly, microsomal lipid peroxidation and oxygen consumption induced by Fe(III)-NTA/AsA were inhibited by the addition of SH-Pal in a time and dose dependent fashion, but GSH and cysteine exhibited a lower protective action. Time course studies on TBARS formation and oxygen consumption indicated the ability of SH-Pal to inhibit initiation and propagation reactions. Moreover, the microsomal lipid peroxidation induced by Cumene hydroperoxide (CumOOH) was progressively suppressed by the addition of increasing amounts of SH-Pal.These findings suggest that the antioxidant action of SH-Pal is partly due to complete reduction of iron at a faster rate and inhibition of oxygen consumption during the progress of the peroxidation. Further, SH-Pal has a protective action against free radical damage by hydroperoxy radical.
Because allelotype analysis of many tumors has been important in the identification of new tumor suppressor genes, here we have analyzed hepatocellular carcinomas (HCCs) derived from F1 hybrid mice between C3H and MSM in detail. The analysis showed no allelic loss in primary HCCs, while the loss was detected in tumor cell lines established from HCCs. Recently, a candidate tumor suppressor gene termed p16/CDKN2, which was located near the interferon gene cluster on human chromosome 9p21, was identified by virtue of its frequent homozygous deletion in cell lines derived from many different tumor types. Since frequent allelic imbalances in the D4MIT9 locus and loss of heterozygosity in the alpha-interferon gene which was located near the mouse homolog of p16/CDKN2 (mouse p16) gene were detected in tumor cell lines, we investigated homozygous deletion of the mouse p16 gene by the comparative multiplex PCR method. The analysis revealed frequent homozygous deletion of the gene in thirteen of the tumor cell lines (13/25, 52%), but not in primary HCCs (0/25, 0%). These data indicate that gene deletions including the mouse p16 gene on chromosome 4 in tumor cell lines occur during the culture and that allelic imbalances are uncommon in mouse primary HCCs. Our results suggest that mouse p16 plays an important role in mouse hepatocarcinogenesis in vivo in progression or immortalization in vitro.
The preventive effects of two antioxidants, methyl 9(or 10)-hydroxy-10(or 9)-mercaptostearate (SH-S) and hexadecanethioic S-acid (thiopalmitic acid, SH-Pal) against the oxidative modification of low density lipoproteins (LDL) induced by cupric ion or a water soluble initiator of peroxyl radicals, 2, 2-azobis(2-amidinopropane) dihydrochloride (AAPH), were studied by measuring thiobarbituric acid-reactive substances (TBARS). SH-S acted as an effective antioxidant in the oxidative modification of LDL induced by either cupric ion or AAPH. Interestingly, SH-S completely inhibited the formation of fluorescence products and decreased both the fluorescence and α-tocopherol content in LDL induced by cupric ion, and reduced 1, 1-diphenyl 2-picrylhydrazyl (DPPH) used as a stable free radical model. The antioxidative effect was effectively prevented by the addition of increasing amounts of N-ethylmalemide (NEM) to the system. SH-Pal also inhibited the cupric ion-induced LDL oxidation, but showed little inhibitory effect on the AAPH-induced LDL oxidation. Moreover, SH-Pal was reduced to palmitic acid during the AAPH-induced LDL oxidation.These findings indicate that SH-S protects against oxidative damage of LDL in vitro, and that it acts as a free radical in peroxidation. In addition, this study shows that SH-Pal doesn't act as an efficient antioxidant in AAPH-induced lipid peroxidation.
By confocal fluorescence microscopy we have studied the rises of the intracellular free calcium ion concentration ([Ca2+]i) in helper T cells (KLH-specific, I-Ak-restricted Th1 cells, 28-4) after interaction with antigen-specific and antigen-nonspecific B cells (antigen-presenting cells). Antigen-specific and antigen-nonspecific B cells were prepared by the preincubation of TNP (trinitrophenol)-specific B cell hybridomas (TP67.21 I-Ak) with TNP-conjugated KLH and KLH alone, respectively. Calcium signals in Th1 cells (28-4) were induced by antigen-specific B cells one hundred times more efficiently as those by antigen-nonspecific B cells. Herbimycin A, a tyrosine kinase inhibitor, suppressed the former signals but not the latter. These results indicated that tyrosine phosphorylation was involved in the antigen processing of antigen-specific B cells but not in the processing of antigen-nonspecific B cells.
Bifidobacterium sp. strain SEN was isolated and characterized by hydrolytic conversion of sennosides to sennidins (Akao et al., Appl. Environ. Microbiol., 60, 1041 (1994)). The sennoside-hydrolyzing capacity of the strain SEN was disappeared following the addition of glucose to the media in spite of good bacterial growth and potent activity hydrolyzing p-nitrophenyl β-D-glucopyranoside (pNPG). In a fructose-containing medium, no such suppressing effect was shown. Following a 10 h incubation in 50 mM potassium phosphate buffer (pH 7.4), the sennoside-hydrolyzing activity of the bacterium increased, dose-dependently, with the addition of sennoside B. Inhibition of the substrate-induced increase in sennoside-hydrolyzing activity was observed following the addition of some antibiotics (chloramphenicol, streptomycin, and rifampicin). In particular, chloramphenicol completely inhibited the increase of sennoside-hydrolyzing activity while 38% pNPG-hydrolyzing activity remained. It is suggested that the strain SEN produces two different β-glucosidases of which the sennoside-hydrolyzing enzyme is inducible. In addition, the glucosides pNPG, esculin, salicin, or amygdalin stimulated the induction of the sennoside β-glucosidase, but less markedly than sennoside. Sennidin A or sugars (glucose, fructose, cellobiose, or maltose) did not induce the enzyme.
A novel β-glucosidase, which is inducible and capable of catalyzing the hydrolysis of sennosides, was purified from Bifidobacterium sp. strain SEN with Triton X-100 solubilization and DEAE-cellulose column chromatography, by which hydrolytic activities toward sennoside B, 4-methylumbelliferyl β-glucoside (MUG), and p-nitrophenyl β-glucoside (pNPG) were obtained together in the same eluted fractions. The activity was stable against detergents such as sodium dodecyl sulfate (SDS) and Triton X-100, but was denatured by SDS and β-mercaptoethanal when heated. The final preparation was shown to be nearly homogeneous on SDS-polycrylamide gel electrophoresis (PAGE) either after the enzyme was denatured or when it was not denatured. In the non-denaturing SDS-PAGE, a single protein band hydrolyzed MUG on the gel. In the denaturing SDS-PAGE, the subunit mass of the enzyme was estimated to be 110 kDa.The enzyme was optimally active at pH 6.0 for hydrolysis of sennoside B and MUG. Km values for sennoside B and MUG are 0.94 and 0.53 mM, respectively. The enzyme also catalyzed the hydrolysis of pNPG, amygdalin, geniposide and salicin. It was less active against methyl β-glucoside and incapable of hydrolyzing cellobiose. The β-glucosidase activity was inhibited by deoxynojirimycin and p-chloromercuribenzenesulfonic acid, but was less susceptible to several metals (FeSO4, ZnCl2, and CuSO4), and 5, 5'-dithio-bis(2-nitrobenzoic acid).
The purpose of this study was to evaluate the accuracy of the recommended theophylline therapeutic range in the treatment of acute airway obstruction. Twenty seven patients (20 to 64 years) with acute asthma attack were given aminophylline intravenously to obtain a theophylline concentration between 10 and 20 μg/ml. Peak expiratory flow rates (PEFR) and serum theophylline concentrations were measured before and after aminophylline injection. When a marked improvement was not seen after aminophylline injection, the treatment was followed by inhalation of a β-agonist and intravenously administered hydrocortisone. In order to clarify the relationship between theophylline efficacy at a therapeutic level and PEFR, as measured before aminophylline administration, the patients were classified into four groups. Group A (n=7) : asthma attack persisted regardless of treatment with aminophylline, β-agonist and hydrocortisone, group B (n=7) : asthma attack improved by aminophylline, β-agonist and hydrocortisone, group C (n=6) : asthma attack improved by both aminophylline and β-agonist, group D (n=7) : asthma attack improved by intravenous aminophylline alone. The means (±S.E.) PEFR before aminophylline administration were 94.3±11.3 l/min in group A, 114.3±10.0 l/min in group B, 196.7±22.2 l/min in group C, and 220.0±12.5 l/min in group D, respectively. There were significant differences in PEFR between the A and C, A and D, B and C, and B and D groups. These findings suggest that theophylline efficacy is not expected in patients with low PEFR (less than 200 l/min) at the time of treatment of an attack, even if a therapeutic theophylline concentration was obtained.
The peroxidation of lipids and changes in the activities of related enzymes in the gastric mucosa were studied in a rat model of gastric mucosal injury induced by the nonsteroidal anti-inflammatory drug indomethacin. The area of gastric erosion and the amount of thiobarbituric acid reactive substances (TBARS) in gastric mucosa were significantly increased beginning 4 h after administration of indomethacin. Xanthine oxidase (XOD) activity in the gastric mucosa also increased immediately after administration of the drug. Although XOD activity was significantly suppressed by allopurinol treatment, the induction of gastric mucosal injury and the increase of TBARS in the gastric mucosa were not. Myeloperoxidase (MPO), a marker enzyme of leukocytes, was unaffected by indomethacin administration. But the depletion of polymorphonuclear leukocyte (PMN) counts induced by an injection of anti-rat PMN antibody inhibited both the injury and the increase in TBARS. Indomethacin activated PMN in peripheral blood at 30 mg/kg per os and enhanced release of oxygen radicals from PMN in peripheral blood. As compared with the XOD system, the generation of oxygen free radicals may derived mainly from activated PMN. On the other hand, superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were reduced by the administration of indomethacin. Decreases in SOD and GSH-px activity in gastric mucosa may aggravate mucosal injury by free radicals and lipid peroxidation.
Ultraviolet radiation is known to induce skin cancer. The induction of DNA damage caused by UV-B and UV-C was investigated using cultured L-132 cells. DNA strand breaks assayed by the alkaline elution procedure occurred in a dose-dependent manner, the extent of the strand breaks were inversely well correlated with the number of viable L-132 cells after 24 h incubation. About a 10-fold dose of UV-B irradiation was required to induce a similar degree of strand breaking to that induced by UV-C. Similarly about a 10-fold dose of UV-B was required to produce a similar amount of pyrimidine dimers, such as cyclobutane-type dimers and pyrimidine-(6-4)-pyrimidone photoproducts, which were determined by ELISA using the specific monoclonal antibody, to that produced by UV-C. Strand breaks induced by UV-B, however, were not fully repaired in viable cells remaining after incubation of cells for a longer period of time, although UV-C-induced strand breaks were repaired in a time-dependent manner. Furthermore, an experiment with a cell-free system, where the induction of strand breaks by repair enzymes did not take place, indicated that UV-B caused significantly more direct DNA strand breaks than that caused by one-tenth the dose of UV-C. The data shown here suggest that UV-B-induced DNA damage is mediated, at least in part, via a different mechanism from the UV-C induced one.
Inspection of the chemical structures of tricyclic antidepressant drugs indicates that they might interfere with the synthesis of thyroid hormones. This iatrogenic potential was demonstrated in vitro by the spectrophotometric detection in both the visible and UV regions of the formation of a complex between antidepressants and iodine. The values of Kc, the formation constant of the drug-iodine complex, were calculated. The concentration of antidepressant which led to a 50% inhibition (IC50) of horseradish peroxidase was also determined. The anti-thyroid activity of drugs can be evaluated from these two parameters, Kc and IC50. The results were compared to those obtained with methimazole, a reference anti-thyroid agent. Antidepressants derived from imipramine appeared to have anti-thyroid activity. This result is now awaiting confirmation in animal experiments.
The effect of an inhibitor of pyrimidine nucleoside phosphorylase (PyNPase), acyclothymidine (AcyT), on the pharmacokinetics of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-FU) was investigated in an oral co-administration of 5'-DFUR and AcyT in rats. AcyT increased the maximal plasma concentration (Cmax) and apparent absorption rate constant (ka) of 5'-DFUR, as expected, but the increase in AUC (area under the curve) was not significant. It was expected that AcyT would only inhibit the phosphorolytic degradation of 5'-DFUR to 5-FU, but the effect was more evident on the pharmacokinetic parameters of 5-FU than on those of 5'-DFUR. AcyT also increased AUC and Cmax of 5-FU when orally co-administered with 5-FU. An inhibitory effect of AcyT on the enzymatic degradation of 5-FU in rat liver and intestinal extract was investigated. AcyT inhibited the degradation in intestinal extract but not in the liver. The result suggests that orally administered AcyT affects the pharmacokinetics of 5-FU partly by inhibiting 5-FU degradation in the process of intestinal absorption as well as by acting as an inhibitor of PyNPase.
This study was conducted to determine the effect of clarithromycin (CAM) on the bioavailability of cyclosporin (CYA) in rats, and to compare its effect with that of erythromycin (EM). The area under the blood CYA concentration-time curve (AUCi.v.) values after intravenous administration of CYA (2 mg/kg) in combination with CAM or EM (100 mg/kg, p.o.) were significantly increased compared with those of CYA alone, suggesting that there was metabolic inhibition of CYA in the liver by CAM or EM. The time to reach the peak concentration after oral administration of CYA (10 mg/kg) tended to be longer with increasing doses of both CAM and EM (10 and 100 mg/kg, p.o.). Each AUCp.o. value for the CAM or EM coadministration group, except the EM (100 mg/kg) coadministration group (about 77% increase), was comparable to that for the CYA alone group. Both CAM and EM (10 and 100 mg/kg, p.o.) were shown to delay gastric emptying in a dose-dependent manner. The gastric emptying in the grioup treated with CAM (100 mg/kg) was significantly lower than that with EM (100 mg/kg). It is suggested that CAM as well as EM might affect the oral bioavailability of CYA by inhibiting its metabolism and simultaneously by changing the gastrointestinal motility in rats. Thus, caution is recommended when administering CYA concomitantly with CAM to humans.
The effect of pulse parameters (duty and frequency) in a constant direct current iontophoresis on the antidiuretic response (elevation in rat urinary osmotic pressure) of desmopressin acetate (DDAVP) was examined in diabetes insipidus rats. Although antidiuretic response was not affected by frequency, it was induced by a duty of more than 26% and prolonged with increasing duty. A positively relationship between dose and AUC, the area under the osmotic pressure-time curve, was confirmed by intravenous administration of DDAVP, and the AUC induced by the iontophoretic delivery increased with increasing duty. The voltage across rat skin required to maintain a constant current density was investigated. A higher voltage was initially applied rat skin in a higher duty. This was related the prolonged pharmacological response induced by iontophoresis.
To improve the bioavailability of the sparingly water-soluble drug, 1-(3, 4-dimethoxyphenyl)-2, 3-bis(methoxycarbonyl)-4-hydroxy-6, 7, 8-trimethoxynaphthalene (TA-7552), the usefulness of the co-grinding method with D-mannitol was investigated. The co-grinding was performed at various weight ratios of TA-7552 and D-mannitol using a ball mill. The particle size was markedly reduced with increasing amount of D-mannitol. A mixture ratio greater than or equal to 1 : 3 of the drug and D-mannitol produced submicron-sized particles. In dogs, bioavailability increased with increasing amount of D-mannitol. The 1 : 9 coground mixture gave complete absorption, as did a lecithin solution of the drug. Even co-ground powders with lower amounts of D-mannitol provided relatively high bioavailability in comparison with ground drug powder alone of a similar particle size. Further, pharamacological examination using rats indicated that the inhibition of cholesterol absorption was intestified with the reduction of particle size. These findings suggest that the co-grinding method with D-mannitol is useful for enhancing the bioavailability and pharmacological effectiveness of this sparingly water-soluble drug.
Studies on the biosynthetic pathway of acetylenic compounds, 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl]phenol and its related compounds, in cultured cells of Asparagus officinalis L. (Liliaceae) revealed that all of the 17 carbon atoms in their skeletons are supplied by phenylalanine. It is also concluded that p-substituted phenylacetylenic moieties (the C6-C2 unit) in these compounds are derived from C6-C3 shikimate pathway metabolites via phenylalanine. As a working hypothesis concerning the C6-O-C3 unit formation, a spirotetrahydrofuran type intermediate is predicted.
The effects of epidermal growth factor (EGF) and cell density of the apperance of β-adrenergic responses were exmianed in primary cultures of adult rat hepatocytes. The β-adrenergic response was measured as the ability to accumulate cAMP by β2-agonist metaproterenol in monolayers that had been cultured without or with 20 ng/ml EGF. Hepatocytes cultured with EGF at a high ell density (1.0×105 cells/cm2) showed a relatively lower response to 10 μM metaproterenol. In contrast, when cultured at a low cell density (3.3×104 cells/cm2) with EGF, the cells showed a higher response to the β-adrenergic agonist. These responses were blocked by the β-adrenergic antagonist propranolol (10 μM). The β-adrenergic response increased rapidly with culture time. The addition of cycloheximide (5 μM) to the culture abolished the expression of β-adrenergic response. The enhanced β-adrenergic response by 20 ng/ml EGF was partially inhibited by the addition of cytochalasin B (20 μM) to the culture. The cAMP-producing response to metaproterenol (10 μM) was dose-dependently inhibited by the specific α2-agonist UK-14304. Pretreatment of the hepatocytes with pertussis toxin (100 ng/ml) potentiated the β-adrenergic response. These results demonstrate that augmented β-adrenergic responsiveness can be acquired by adult rat hepatocytes cultured with 20 ng/ml EGF at a low cell density, and the β-adrenergic response involves de novo synthesis of protein(s). The results also show that significant α2- and β-adrenergic rsponses coexist in the primary cultures of adult rat hepatocytes.
Liposomes were prepared from hydrogenated lecithin (H-PC) by sonication (S) or injection (I) of H-PC dissolved in ethanol containing dl-tocopherol acetate (VEA). The effects of liposomes on the dermal absorption of VEA were studied. The particle diameter of S-liposomes was smaller than that of I-liposomes. The penetration of liposomal H-PC into the skin was much higher for S-liposomes than for I-liposomes 30 min after application to the arms of healthy human volunteers and also to hairless rat back skin. The penetration of 14C-VEA into hairless rat back skin was higher from the liposomes than from free VEA, and the 14C-VEA penetration was higher from S-liposomes than from I-liposomes. 3H-Dipalmitoylphosphatidylcholine and 14C-VEA, which had been entrapped in liposomes, were not detected in plasma. H-PC inhibited the peroxidation of skin lipids. H-PC enhanced the penetration of VEA into the skin, but the degree of enhancement depended on the size of the liposomes, indicating that this liposomal characteristic was an important factor in dermal absorption and/or penetration.
A highly sensitive and selective liquid chromatographic (HPLC) method with post-column fluorescence detection has been developed for the determination of tryptophan 5-monooxygenase activity in rat brain tissue homogenate. 5-Hydroxytryptophan, formed enzymatically from tryptophan (incubation time, 20 min), is extracted with perchloric acid and determined by HPLC. Detection is performed fluorometrically after post-column derivatization by a reaction with benzylamine in the presence of potassium hexacyanoferrate (III). The lower limit of detection for 5-hydroxytryptophan formed enzymatically is 100 fmol at a signal-to-noise ratio of three.
Differences in the ability of metabolically inert peroxisome proliferators [perfluoro-n-decanoic acid (PFDA, C10), perfluoro-n-octanoic acid (PFOA, C8), perfluorooctane sulfonic acid (PFOS, C8) and 1H, 1H-pentadecafluoro-n-octanol (PFOL, C8)] to induce liver microsomal carboxylesterase RL4 in male rats were studied by evaluating changes in the RL4 content by immunoblot analysis with a specific antibody. The administration of PFOA, PFOS and PFOL markedly increase the content of carboxylesterase RL4. On the other hand, PFDA decreases PNPA, BUTA, and ISOC hydrolase activity, and slightly increases the carboxylesterase RL4 content.
We studied the susceptibility of the pyrrolidone moiety and the pyroglutamyl-peptide bond at pGlu-X-Ala-Phe-OH (X=Gly, Ala, Tyr, Ile, Pro, His, Lys, Arg, Thr, Ser, Asp, Glu and Trp) to 1 N HCl or 2 M trifluoromethanesulfonic acid at 60°C. Here we describe the rates of the cleavage reaction of the pGlu-X bond, the pyrrolidone ring-opening reaction of the pGlu moiety and the hydrolysate accumulation. The rank order of the susceptibility rates of the cleavage reactions was Ser>Pro, Gly>Arg, Ala, Glu, Thr, Asp>His, Lys>Trp, Tyr, Ile, and that of the ring-opening reaction was Ile>Tyr, Trp>Arg, His, Lys, Asp>Glu>Ala>Pro, Gly>Ser>Thr. The rank order of the half-lives of the model peptides was Pro>Arg, Lys, Ile>His, Glu>Ala, Tyr>Asp>Gly>Ser>Thr. The results indicated that a bulky and sterically hindered side chain of the amino acid residue neighboring the pGlu moiety favors the ring-opening reaction, and retards the decomposition on acid hydrolysis and the cleavage reaction. Thus, the ring-opening and the cleavage reactions were greatly affected by the amino acid residue neighboring the pGlu moiety in the hydrolysis of pGlu-peptides.
The effects of polycarbophil on the absorption of various nutrients were evaluated by several in situ methods. Polycarbophil reduced the absorption of 3-O-methyl-D-glucose (3-OMG) and L-phenylalanine in the in situ loop and the in situ perfusion methods, but it did not affect the absorption of these nutrients in an open system, the in situ modified loop method, which is closer to physiological conditions. It also did not affect the absorption of vitamin A or phosphatidylcholine-L-α-dipalmitoryl in the latter system. These results indicate that the absorption of nutrients is probably not altered by polycarbophil under physiological conditions.
Macromolecular conjugates of mitomycin C (MMC) were sunthesized by binding an active ester of glutarylated MMC (MMC-G-OSu) to human holo-transferrin (TF). Water-soluble TF-MMC conjugates (TF-G-MMC) were obtained in a good yield (>95%) by this method. The MMC content of the conjugate increased (0.82-9.49 MMC/w%) with increasing amounts of MMC-G-OSu added to the conjugation mixture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed no aggregation in these conjugates. 125I-TF-G-MMC was bound specifically to the TF receptor on Sarcoma 180 cells; the measurement of equilibrium binding of the 125I-labeled conjugate resulted in a saturation isotherm. The amount of conjugate specifically bound to the TF receptor decreased as the MMC content of the conjugate increased. However, it was found that the conjugate with an MMC content below 10 mol MMC/mol TF still retains a binding activity of more than half that of TF. Therefore, when an optimal chemical modification was chosen, TF could be used as a tumor specific drug carrier.