[Development of HIV protease inhibitors as anti AIDS drugs: molecular design based on substrate transition state].
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Amino Acid Sequence↗
Biomedical subjects
Y Kiso
Publications and source records attributed to Y Kiso.
Gicerin, a cell adhesion molecule, participates in the histogenesis of retina.
Gicerin is a novel cell adhesion molecule that belongs to the immunoglobulin superfamily. Gicerin protein adheres to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family, and also exhibits homophilic adhesion. Heterophilic adhesion of gicerin to NOF is thought to play an active role in neurite outgrowth of developing retinal cells in vitro. In this study, we examined the adhesion activity of gicerin during the retinal development of Japanese quail using an antibody directed against gicerin, to elucidate the biological importance of gicerin in retinal histogenesis. Immunohistochemical and Western blot analysis showed that gicerin was highly expressed in the developing retina but suppressed in the mature retina. The aggregation of neural retinal cells from 5-day embryonic quail retina was significantly inhibited when incubated with a polyclonal antibody to gicerin, suggesting that gicerin protein participates in the adhesion of neural retinal cells of the developing retina. Furthermore, histogenesis of retina both in the organ cultures and in ovo embryos was severely disrupted by incubation with a gicerin antibody. These findings provide evidence that gicerin plays an important role in retinal histogenesis.
Animals↗
[Clinical study on subacute thyroiditis and painless thyroiditis].
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Anti-Inflammatory Agents↗
Substituted 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-dione derivatives as novel nonpeptide inhibitors of human heart chymase.
A series of 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-dione derivatives have been synthesized and evaluated for their ability to selectively inhibit human heart chymase. The structure-activity relationship studies on these compounds gave the following results. The 1-phenyl moiety participates in a hydrophobic interaction where an optimum size is required. At this position, 3,4-dimethylphenyl is the best moiety for inhibiting chymase and showed high selectivity compared with chymotrypsin and cathepsin G. A 3-phenylsulfonyl moiety substituted with hydrogen-bond acceptors such as nitrile and methoxycarbonyl enhances its activity. Molecular-modeling studies on the interaction of 3-[(4-chlorophenyl)sulfonyl]-1-(4-chlorophenyl)-imidazolidine-2,4-dione (29) with the active site of human heart chymase suggested that the 1-phenyl moiety interacts with the hydrophobic P1 pocket, the 3-phenylsulfonyl moiety resides in the S1'-S2' subsites, and the 4-carbonyl of the imidazolidine ring and sulfonyl group interact with the oxyanion hole and the His-45 side chain of chymase, respectively. The complex model is consistent with the structure-activity relationships.
Binding Sites↗
Effects of maternal bilateral ureteral ligation on formation of the glomerular basement membrane in fetal rat kidney.
BACKGROUND: The present study was designed to clarify development of filtration property of fetal renal glomerulus when maternal kidney is dysfunctional. MATERIALS AND METHODS: Maternal bilateral ureteral ligation was performed on days 17, 19, and 21 of pregnancy. One day after each operation, cationized ferritin (CF), native ferritin (NF), and horseradish peroxidase (HRP) were injected, respectively, in the fetuses. Distribution of the tracers in the fetal kidneys was investigated electron microscopically. RESULTS: On fetal day 20, clustered CF particles were present in the laminae rarae interna and externa of the glomerular basement membrane of the fetuses from ureter-ligated mothers, while the clusters were arrayed in three to four layers in that of the fetuses from sham-ligated ones. On fetal day 22, a relatively large amount of CF particles was present in the lamina rara externa in the fetuses from ureter-ligated mothers when compared to that in the age-matched control fetuses. On fetal days 20 and 22, the number of NF particles was decreased, and shortening of the time for filtration of HRP through the glomerular basement membrane was observed in the fetuses from the ligated mothers. CONCLUSIONS: These results suggest that dysfunction of maternal kidneys causes accelerated formation of fetal glomerular basement membrane and stimulates glomerular function in filtration in fetal rat kidney.
Animals↗
Characterization of the cells that migrate from metrial glands of the pregnant mouse uterus during explant culture.
Granulated metrial gland (GMG) cells are estrogen-receptor and Interleukin 2 (IL-2) receptor positive lymphocytes of the Natural Killer cell lineage found in the murine uterus during pregnancy. Functional studies of these cells, which are now more frequently called uterine NK (uNK) cells, have been limited due to technical difficulties. The cells are difficult to isolate and their proliferation and differentiation have not been achieved in culture. In 1988, Mukhtar and Stewart (Cell Tiss. Res., 253, 413-417) reported a method for explant culture of metrial glands isolated from pregnant rodents that yielded an almost pure population of uNK cells. This major technical advance has supported most of the subsequent functional and molecular studies of rodent uNK cells. However, the quality of the cells isolated by the explant culture procedure has not been established. A cytochemical approach was used to identify and quantify the cells migrating from metrial glands. At midpregnancy, almost all (> 90%) migrating nucleated cells were NK cells. Earlier in gestation, a significant proportion (25%) of cells having lymphoid morphology could not be assigned to the lineage. The viability of cells migrating from explants was assessed by DNA isolation and electrophoresis on days 6-16 of gestation. At all times evidence for apoptosis was found, even after culture intervals as brief as 4 h. Parallel analyses of histological sections of the metrial gland, using terminal deoxytransferase labelling to detect nuclear fragmentation, did not support significant levels of uNK cell death in situ prior to day 12 of gestation. Supplementation of the explant culture medium with estrogen, IL-2, various extracellular matrices, decidual cells or combinations of these did not lead to in vitro proliferation of uNK cells and usually did not extend the short term viability of these cells in serum supplemented or serum free media. Thus, the optimal culture conditions for uNK cells remain undefined.
Animals↗
The development of transient hypothyroidism after iodine-131 treatment in hyperthyroid patients with Graves' disease: prevalence, mechanism and prognosis.
OBJECTIVE: Recovery of thyroid function in patients following hypothyroidism induced by 131I therapy for Graves' disease has been described, but only a few detailed clinical and biochemical studies of this phenomenon (transient hypothyroidism) have been published. The prevalence, mechanism, and final outcome of transient hypothyroidism in 260 patients with Graves' disease treated with 131I was studied. DESIGN: A retrospective study. PATIENTS: Two hundred sixty patients with Graves' disease, treated with 131I between 1 and 15 years previously, were categorized into 4 groups according to their thyroid function during and 1 year after therapy (Group 1: permanent hypothyroidism, 28 patients; Group 2: transient hypothyroidism, 39 patients; Group 3: euthyroidism without transient hypothyroidism, 83 patients; Group 4: hyperthyroidism, 110 patients). MEASUREMENTS: We compared total T4, total T3, TSH, anti-thyroglobulin (TGHA) and anti-microsomal (MCHA) antibodies, the TSH-binding inhibitory immunoglobulin (TBII) index, thyroid weight, dose of 131I, and 24-hour 131I uptake as pretreatment variables. The mean time for permanent hypothyroidism to develop was estimated by the Kaplan-Meier product limit method. The TBII index and thyroid stimulating antibody (TSAb) activity were measured in seven patients from Group 1 and in nine patients from Group 2 at the time that they became hypothyroid. RESULTS: Hypothyroidism developing within 12 months of therapy was transient in 58% (39/67) of patients. No pretreatment variables were found to differ between patients with and without transient hypothyroidism. The mean estimated time between therapy and the development of permanent hypothyroidism was 96 months in Group 2; this time interval was significantly shorter than 126 months in Group 3 and 129 months in Group 4 (P < 0.05, P < 0.01, respectively). TSAb activity was > 500% In 78% (7/9) of patients from Group 2, which was significantly higher than that found (14%, 1/7) in Group 1. CONCLUSIONS: These results indicate that (1) more than half the patients who developed hypothyroidism within 6 months after 131I treatment for Graves' disease recovered spontaneously, (2) TSAb activity might play some role in the recovery of transient hypothyroidism, and (3) the development of transient hypothyroidism may influence long-term thyroid function.
Adult↗
Growth hormone-releasing hormone (GHRH)-GH-somatic growth and luteinizing hormone (LH)RH-LH-ovarian axes in adult female transgenic mice expressing human GH gene.
We have examined alterations in the hypothalamo-pituitary GH-somatic growth axis and the hypothalamo-pituitary LH-ovarian axis in a line of transgenic ICR mice expressing human GH (hGH) under the influence of the whey acid protein promoter. Transgenic female mice weighed twice as much as control females and were infertile. The size of the anterior pituitary (AP) was 1/3 that of the controls. In transgenic mice, acinar cells in the mammary and mandibular glands displayed hGH-immunoreactivity, and plasma hGH was detected by radioimmunoassay. In the medial basal hypothalamus (MBH) of transgenic females, the immunoreactive-GHRH level was decreased (P<0.01). There was a corresponding reduction in the number of GHRH-immunoreactive neurons in the arcuate nucleus (ARC) and in the immunostaining of GHRH nerve terminals in the median eminence. The level of somatostatin (SRIH) in the MBH was increased (P<0.05), and SRIH-immunoreactive neurons in the periventricular nucleus (PeV) were increased in size and number in transgenic mice. The MBH level of LHRH in transgenic animals was greater (P<0.01) than in controls, although there was no apparent difference in the number of LHRH-immunoreactive neurons or in LHRH level in the preoptic area. There are fewer SRIH- and LHRH-immunoreactive neurons in the ARC in transgenic mice. Cells in the AP for GH, PRL, and LH were fewer in transgenic mice. The ovary suffered disturbance of follicular development and of corpora lutea formation. These results demonstrate that chronic overproduction of hGH may profoundly affect the organization of the GHRH/SRIH-GH-somatic growth axis and the LHRH-LH-ovarian axis due to reduction of GHRH-, SRIH- and LHRH-neurons in the ARC and increase of SRIH-neurons in the PeV.
Animals↗
A reductive acidolysis final deprotection strategy in solid phase peptide synthesis based on safety-catch protection.
A reductive acidolysis final deprotection strategy in solid phase peptide synthesis was developed using a new safety-catch type of semi-permanent protecting groups and new linkers which were derived from 4-methylsulfinylbenzyl protection. This new strategy was based on a two-dimensional protection scheme employing acid-labile temporary and acid-stable but reductive acidolysis-cleavable semi-permanent protecting groups. By using this strategy, we successfully synthesized four model peptides, of which two contained C-terminal amide.
Amino Acid Sequence↗
A study of granulated metrial gland cells in the pregnant, alymphoplasia (aly/aly) mice.
During pregnancy, a population of uterine NK cells, commonly called granulated metrial gland (GMG) cells, differentiates in the uterus of both immune competent and various immunodeficient mice. Regulatory mechanisms controlling the differentiation of GMG cells are not fully known. It has been proven that GMG cells are derived from bone marrow, appear under the influences of progesterone and estrogen, do not require the presence of an embryo, and are associated in rodents with decidualization of the uterine stroma. Mice of genotype aly/aly are genetically deficient in lymph nodes and Peyer's patches due to a lymphoid-associated mesenchymal disorder. They are considered to be a useful model for the study of interactions between lymphocytes and stromal components. This immunodeficient animal is completely different from nu/nu and scid/scid mice who differentiate GMG cells during pregnancy. To determine whether the differentiation of GMG cells depends on mesenchymal interactions in the uterus, aly/aly mice were studied histologically between days 10 and 14 of pregnancy for differentiation of GMG cells and development of the metrial gland. Metrial gland tissue was present and appeared normal in aly/aly mice. There were no significant differences in the distribution of GMG cells in comparison to control pregnant aly/+ mice. Fewer GMG cells were present in aly/aly mice than aly/+ mice on days 12 and 14 of pregnancy. The features of individual GMG cells were different on days 10 and 12 of pregnancy. GMG cells in aly/aly mice were small in size and the granules were poorly developed. By day 14, however, GMG cells acquired a mature size and the granules appeared mature. It is likely that GMG cell differentiation was delayed in pregnant aly/aly mice, due to a mesenchymal disorder affecting metrial gland development in this animal.
Animals↗
A case of a pony with Cushing's disease.
The pars intermedia of the pituitary gland, and plasma ACTH and cortisol levels in the pony, which was first diagnosed in Japan as indicating equine Cushing's disease, were examined by immunohistochemistry and radioimmunoassay, respectively. The pars intermedia was greatly enlarged and most of its cells were immunoreactive for antisera to both adenocorticotropic hormone (ACTH) and beta-endorphin (beta-End). The plasma ACTH level was elevated when clinical symptoms appeared. The present results reveal that equine Cushing's disease in this pony was induced by the hypersecretion of ACTH and beta-End from the enlarged pars intermedia of the pituitary gland.
Adrenal Glands↗
[Approval of HIV protease inhibitors as the AIDS therapeutics of next generation].
The long-term usages of reverse transcriptase inhibitors as anti-HIV drugs have induced serious side effects and emergence of resistant viruses. To overcome these problems, combination therapies are expected to be effective. Under such circumstances, HIV protease inhibitors have been rationally designed and synthesized based on the substrate transition state concept. HIV protease inhibitors act at the exact point in the life cycle of HIV, exhibited efficacy in the clinical trials and have been approved with high expectation. The introduction of anti-HIV drug with novel action mechanism evoked a new dramatic movement in the combination therapy of AIDS.
Amino Acid Sequence↗
Immunocytochemical localization of adrenocorticotropic hormone-immunoreactive cells of the pars intermedia in thoroughbreds.
OBJECTIVE: To analyze and characterize adenoma and hyperplasia of the pars intermedia (PI) of Thoroughbred mares. ANIMALS: 165 Thoroughbred mares, without clinical signs of hyperadrenocorticism that had been euthanatized or had died, of causes such as sudden death, colic, pneumonia, or trauma, and were necropsied. Five of those horses, 17 to 25 years old, had a large pituitary gland at necropsy. Eight mares, 5 to 15 years old with normal-size pituitary gland, were selected at random for comparison. PROCEDURE: A morphologic comparison of the pituitary gland between horses with and without tumors of the PI was conducted by use of immunocytochemistry and morphometry. RESULTS: In horses with normal pituitary gland, the PI was supplied by a vast capillary or sinusoidal plexus, which connected that in the pars distalis (PD) with that in the pars nervosa (PN). Cells of the PI stained slightly with ACTH antiserum, but some cells in the border region, which is contiguous to the PD, were strongly ACTH immunoreactive. At necrospy, horses with an enlarged pituitary gland also had adenoma of the pituitary gland involving the PI. Cells of the border region were hypertrophied and stained strongly with ACTH antiserum. The area and number of individual tumor cells of the border region of the PI of horses with adenoma were significantly increased, compared with those in horses with normal pituitary gland. CONCLUSIONS: Cells of the PI-PD border region may secrete substantial quantities of ACTH, owing to stimulation by corticotropin-releasing factor. Adenoma and hyperplasia of the PI in Thoroughbred mares may be associated with hyperadrenocorticism.
Adenoma↗
[Structural analysis of protease-inhibitor system using graphics].
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Amino Acid Sequence↗
Analysis of pesticides in environmental water samples by solid-phase micro-extraction-high-performance liquid chromatography.
Solid-phase micro-extraction (SPME) followed by high-performance liquid chromatography (LC) has been applied to analyze pesticides in water samples. A device interfaces SPME to the LC injection process by solvent extracting analytes from the fiber and then introducing the solvent into the LC injector. LC analysis with UV detection was performed first with a conventional column (4.6 mm I.D.). To enhance efficiency of SPME, three extraction conditions, stirring, temperature and salt concentration, were optimized. Subsequently, semi-microcolumn LC (1.5 mm I.D.) was evaluated for this method, giving lower detection limits and less solvent consumption. Detection limits were within Japanese regulatory limits for drinking water. The SPME-LC method was applied to real-world environmental water samples.
Acetonitriles↗
Solution NMR evidence that the HIV-1 protease catalytic aspartyl groups have different ionization states in the complex formed with the asymmetric drug KNI-272.
In order to improve the design of HIV-1 protease inhibitors, it is essential to understand how they interact with active site residues, particularly the catalytic Asp25 and Asp125 residues. KNI-272 is a promising, potent HIV-1 protease inhibitor (K(i) approximately 5 pM), currently undergoing phase 1 clinical trials. Because KNI-272 is asymmetric, the complex it forms with the homodimeric HIV-1 protease also lacks symmetry, and the two protease monomers can have distinct NMR spectra. Monomer specific signal assignments were obtained for amino acid residues in the drug binding site as well as for six of the eight Asp residues in the protease/KNI-272 complex. Using these assignments, the ionization states of the Asp carboxyl groups were determined from measurements of (a) the pD dependence of the chemical shifts of the Asp carboxyl carbons and (b) the H/D isotope effect upon the Asp carboxyl carbon chemical shifts. The results of these measurements indicate that the carboxyl of Asp25 is protonated while that of Asp125 is not protonated. These findings provide not only the first experimental evidence regarding the distinct protonation states of Asp25/125 in HIV-1 protease/drug complexes, but also shed light on interactions responsible for inhibitor binding that should form the basis for improved drug designs.
Amino Acid Sequence↗
Design and synthesis of substrate-based peptidomimetic human immunodeficiency virus protease inhibitors containing the hydroxymethylcarbonyl isostere.
The human immunodeficiency (HIV) codes for an aspartic protease known to be essential for retroviral maturation and replication. The HIV protease can recognize Phe-Pro and Tyr-Pro sequences as the virus-specific cleavage site. These features provided a basis for the rational design of selective HIV protease-targeted drugs for the treatment of acquired immunodeficiency syndrome (AIDS). HIV protease is formed from two identical 99 amino acid peptides. We replaced the two Cys residues by L-Ala to synthesize [Ala67,95]-HIV-1 protease by the solid phase method and then prepared [Tyr6,42, Nle36,46, (NHCH2COSCH2CO)51-52, Ala67,95] HIV-1 protease (NY-5 isolate) using the thioester chemical ligation method. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing an unnatural amino acid, (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) with a hydroxymethylcarbonyl (HMC) isostere. Among them, the conformationally constrained tripeptide kynostatin (KNI)-272 (iQoa-Mta-Apns-Thz-NHBut) was a highly selective and superpotent HIV protease inhibitor (Ki = 0.0055 nM). KNI-272 exhibited potent antiviral activities against both AZT-sensitive and -insensitive clinical HIV-1 isolates as well as HIV-2 with low cytotoxicity. After i.d. administration, bioavailability of KNI-272 was 42.3% in rats. Also, KNI-272 exhibited in vivo anti-HIV activities in human PBMC-SCID mice. The x-ray crystallography and molecular modeling studies showed that the HMC group in KNI-272 interacted excellently with the aspartic acid carboxyl groups of HIV protease active site in the essentially same hydrogen-bonding mode as the transition state. This result implies that the HMC isostere is an ideal transition-state mimic and contributes to the high activity of KNI-272.
Acquired Immunodeficiency Syndrome↗
The bioavailability of oral dosage forms of a new HIV-1 protease inhibitor, KNI-272, in beagle dogs.
The bioavailability (BA) of a tripeptide protease inhibitor, KNI-272, which has a strong pharmacological potential for treating human immunodeficiency virus type 1 (HIV-1), has been studied in beagle dogs by administering several oral dosage forms. The tested dosage forms were form 1, plain gelatin capsules; forms 2 and 3, gelatin capsules of which the inner and outer surfaces were coated with 7G ethylcellulose (EC, 30 mu m thickness) and an enteric coating material, hydroxypropyl methylcellulose phthalate (HP-55), respectively; and form 4, gelatin capsules of which the inner surface is coated with 10G EC (60 mu m thickness). The difference between forms 2 and 3 was the amount of citric acid contained in the capsule, namely 100 mg in form 2 and 200 mg in form 3. One hundred milligrams of KNI-272 was placed in each capsule after being dissolved with propylene glycol (PG). These capsules were used to deliver KNI-272 to the stomach for form 1, to the upper part of the small intestine for forms 2 and 3, and to the middle part of the small intestine for form 4. As a reference, 50.0 mg of KNI-272 was administered to the same dogs by intravenous (IV) infusion for 15 min. By measuring the plasma drug levels with the HPLC method, BAs were estimated for each test dosage form. Form 1 showed the highest BA of 26 center dot 2 +/- 7 center dot 0% (mean +/- SE), though the other capsules showed BAs of approximately 10%, namely 6 center dot 6 +/- 0 center dot 4% for form 2, 10 center dot 3 +/- 1 center dot 1% for form 3 and 14 center dot 2 +/- 1 center dot 0% for form 4. Therefore, as the site where KNI-272 is released from the capsule becomes higher, the BA increases. In addition, as the amount of citric acid contained in a capsule increases, the BA value tends to increase. These results suggest that KNI-272 is stable and not extensively hydrolysed in the gut after oral administration, that the dissolution process into GI fluids is important for the BA of KNI-272, and that the most appropriate absorption site of KNI-272 in dogs is the duodenum. The potential of this new tripeptide compound as an orally active anti-AIDS drug has been confirmed.
Administration, Oral↗