Biomedical subjects
Publications and source records attributed to H T Lee.
Pachyonychia congenita is characterized by symmetrical nail dystrophy, palmoplantar keratoderma, oral leukokeratosis, and follicular hyperkeratosis. In addition to these features, multiple cutaneous cysts of various kinds have been described. We report a case of pachyonychia congenita associated with eruptive vellus hair cyst.
AIM: To develop nonpeptide tachykinin NK3 antagonists. METHODS: Five tachykinin NK3 antagonists were synthesized. Receptor binding assay and oral absorption study were made. RESULTS: The 4,4-disubstituted piperidine compounds (1b, 1c, and 1d) showed stronger activities (IC50 = 5.9, 6.2, and 11 nmol.L-1, respectively) than the monosubstituted ring compound 1e (IC50 = 17 nmol.L-1). 4-Phenyl (1b) and 4-phenylsulfonylmethyl (1c) compounds were more active than the 4-fluorobenzyl compound (1d). All antagonists were found to be orally absorbable, the T1/2 of 1b (6.4 h) was more than three-fold longer than that of 1a (1.9 h). CONCLUSION: Compound 1b had the best binding activity (IC50 = 5.9 nmol.L-1) and the best AUC (2081 micrograms.h.L-1).
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The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB1-2), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of Calpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.
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Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.
Sulfoacetic acid, phosphoramidate, and phosphoramide analogs of the ACAT inhibitors, CI-999 and CI-1011 were synthesized. The structure-activity relationships of these compounds as ACAT inhibitors are described.
The objective of this study was to determine microtubule assembly and chromatin configuration in porcine oocytes during the first cell cycle following round spermatid injection into matured porcine oocytes in the presence or absence of electrical stimulation. The oocytes with two large pronuclei and two polar bodies were classified as normal fertilization at 6 to 8 h following injection. The incidence of normal fertilization following round spermatid injection with electrical stimulation was significantly higher (21/45, 47%) than that following injection alone (6/39, 15%). Although a small microtubular aster was organized near the decondensed spermatid chromatin in some oocytes (2/6, 33%, spermatid injection alone; 9/21, 29%, spermatid injection and electrical stimulation), it did not enlarge nor fill the cytoplasm. Instead, a dense network of microtubules in the cytoplasm was organized from cortex. At 12 to 15 h after injection, we classified the oocytes with closely apposed pronuclei as normal fertilization. The electrical stimulation following spermatid injection enhanced (P < 0.05) the incidence of normal fertilization (18/54, 33%) compared with spermatid injection alone (7/52, 13%). During pronuclear movement, the maternally derived microtubules filled the whole cytoplasm, which appeared to move male and female chromatin. Mitosis and two-cell division were observed at 20 to 24 h after spermatid injection with electrical stimulation (12/41, 29%). At mitotic metaphase, the microtubular spindle had focused astral poles, and chromosomes were aligned on the spindle equator. During mitosis, asters were assembled at each spindle pole, and they filled the cytoplasm. These results suggested that round spermatid nuclei of the pig can develop into a morphologically normal pronucleus in matured porcine oocytes and are competent to participate in syngamy with the ootid chromatin. In addition, functional microtubules for complete fertilization with spermatid were not associated with male-derived centrosome but were organized solely from maternal stores.
We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n=35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome.
Elevated plasma lipoprotein(a) [Lp(a)] is an independent risk factor for several vascular diseases. Lp(a) particles are generated through the formation of a disulfide bond between Cys4057 of kringle IV type 9, (KIVt9), of the multikringle apolipoprotein(a) [apo(a)] and a cysteine in apoB-100 low-density lipoprotein (LDL). To better understand this interaction, we have expressed and purified KIVt9 from Escherichia coli as a His-Tag fusionprotein. Dithiothreitol (DTT)-treated purified KIVt9 migrated as a single approximately 17. 3-kDa band on SDS-PAGE gels. Without DTT, an additional band twice the molecular weight of KIVt9 was observed. The double-size band presumably resulted from dimerization of individual kringles, through their unpaired cysteine residues, since a mutation Cys4057 --> Ser ([Ser4057]KIVt9) abolished dimer formation. Using a gel-shift assay, we showed that KIVt9 could couple to 14-amino-acid apoB-100 synthetic peptides (apoB3732-3745 and apoB4319-4332) containing Cys3734 or Cys4326. Both of these apoB-100 cysteines have been reported to associate with apo(a) to generate Lp(a). In the presence of either apoB-100 peptide, KIVt9 was shifted to a higher molecular weight that was consistent with the covalent addition of a 1.2-kDa apoB-100 peptide. Identical apoB-100 peptides in which the cysteine residues were replaced by alanine ([Ala3734]apoB3732-3745 and [Ala4326]apoB4319-4332) had no effect in the gel-shift assay. Furthermore, [Ser4057]KIVt9 did not covalently interact with apoB3732-3745 or apoB4319-4332. These results indicated that KIVt9 couples to the Cys-apoB-100 peptides through a disulfide linkage. This system may be suitable for further investigating the apo(a)/apoB-100 coupling reaction and the structure of KIVt9 through X-ray crystallographic studies.
Lipoprotein(a) (Lp(a)), which is generated through the covalent association of apolipoprotein(a) (apo(a)) and apo B-100-LDL, is an independent risk factor for several vascular diseases. Therefore, there is interest in developing therapies for lowering Lp(a). This investigation was carried out to determine the effect of CI-1011, a potent lipid regulator in rodents, on Lp(a) and other lipid parameters in cynomolgus monkeys (Macaca fascicularis). Nine healthy male monkeys on a normal chow diet were orally treated with CI-1011 at 30 mg/kg per day for 3 weeks. Lp(a) and total cholesterol levels were significantly decreased after 1 week and maximally reduced to 68 and 73% of control levels, respectively, after 3 treatment weeks. The decreases in total cholesterol were mainly due to changes in low density lipoprotein (LDL). The LDL:HDL ratio decreased by 30%. Triglycerides were unaffected by treatment. Lp(a) and total cholesterol levels returned to pretreatment values after stopping treatment suggesting a direct effect of the compound on their inhibition. Further studies demonstrated that CI-1011 was effective at a low dose of 3 mg/kg per day after 1 week of administration. CI-1011 also decreased apo B-100 to 80% of control levels, but this change was not sufficient to account for the Lp(a) lowering. There was also no correlation between the changes in Lp(a) and apo B-100 levels. Treatment of cynomolgus monkey primary hepatocyte cultures with CI-1011 resulted in a dose-dependent inhibition of Lp(a) levels suggesting a direct hepatic effect of the compound. Western blot analysis of the samples showed that changes in Lp(a) were associated mainly with decreased apo(a) (47%), but not apo B-100 (17%). These results demonstrate that CI-1011 effectively decreases Lp(a) levels both in vivo and in vitro.
Muscle relaxant pharmacophysiology can be altered in various clinical situations. We report increased requirement of vecuronium in a patient diagnosed with testicular feminization. Increased level of endogenous testosterone and steroidal-core structure of vecuronium may explain the increased tolerance to vecuronium in this patient.
UNLABELLED: To assess the feasibility of manipulating human cerebrovascular resistance with adenosine, we measured cerebral blood flow (CBF) by determining the initial slope (IS) of tracer washout 20-80 s after intracarotid 133Xe injection (standard IS) during sequential 3-min intracarotid infusions of (a) saline; (b) adenosine 1.2-mg bolus followed by an infusion of 1 mg/min (bolus + infusion); (c) saline; and (d) nicardipine (0.1 mg/min). During 133Xe washout, adenosine caused a rapidly clearing compartment. Therefore, tracer washout was also analyzed 5-25 s after injection (early IS). Nicardipine (n = 8) increased both standard IS (from 39+/-12 to 53+/-16 mL 100g.min(-1); P < 0.005) and early IS (from 40+/-9 to 55+/-20 arbitrary units; P < 0.02) to a similar degree. Adenosine bolus + infusion increased early IS (from 33+/-6 to 82+/-43 arbitrary units; P < 0.02) but did not increase standard IS (from 41+/-12 to 43 +/-16 mL 100g(-1) min(-1)). Standard and early IS values were then determined before and after adenosine delivered either by infusion alone (2 mg/min for 3 min, n = 5) or bolus alone (2 mg in 1 s, n = 3). Neither standard nor early IS changed after adenosine infusion alone. Early IS increased after adenosine bolus alone. Increase in early IS, but not standard IS, suggests a transient (<30 s) increase in CBF. IMPLICATIONS: Intracarotid adenosine, in the 1- to 2-mg dose range, may cause a transient, but not a sustained, increase in cerebral blood flow. Intracarotid adenosine in such a dose range does not seem to be an appropriate drug for sustained manipulation of cerebrovascular resistance.
Self-injurious behavior occurring in persons with severe mental retardation is a clinically significant and poorly understood problem. Multiple neurotransmitter systems have been implicated in the pathogenesis of this behavior, particularly dopaminergic, opioidergic, and serotonergic systems. Pemoline, a central stimulant, administered systemically at high doses reliably produces self-biting behavior in the rat. The systemic bolus of pemoline produces sustained neostriatal levels of pemoline for over 24 h in a continuous infusion paradigm. Studies of the effect of cortical lesions on pemoline-mediated behaviors reveal that cortical damage, as is common in profound mental retardation, lowers the threshold for pemoline-induced self-biting behavior. Data from the corticostriatal slice suggests that sustained exposure to pemoline produces a shift in N-methyl-D-aspartate receptor-mediated responses rendering them more susceptible to dopaminergic enhancement. Thus, dopaminergic and glutamatergic interactions appear to play an important role in the development and expression of self-biting in the pemoline model.
The objective of this study was to determine the effects of fetal calf serum (FCS), non-essential MEM amino acids, MEM vitamins and insulin on blastocoel formation, expansion and hatching in porcine embryos developing in vitro. Addition of 20% FCS to the NCSU 23 medium significantly (P < 0.05) decreased by the compaction and blastocoel formation of 1- to 2-cell embryos developing in vitro. In contrast, more 1- to 2-cell embryos commenced hatching in the media containing amino acids than in control medium (25.7 vs 2.6%, P < 0.01). Amino acids and insulin synergistically enhanced the incidence of blastocoel formation and hatching of porcine embryos developing in vitro (P < 0.05). When early compacted embryos which developed in vitro in NCSU 23 medium were cultured in BSA-free NCSU 23 medium supplemented with 20% FCS, the incidence of hatching was significantly increased compared with that of the control groups (35.7 vs 4.1%, P < 0.01). However, addition of amino acids, vitamins or insulin to the NCSU 23 medium did not enhance the development of early morulae to the hatched embryos (P > 0.1). When either in vivo or IVM/IVF-derived 1- to 2-cell stage embryos were cultured 4 d in the modified NCSU 23 and an additional 4 days in the modified NCSU 23 supplemented in the FCS, the percentages (61.8 and 17.8%, in vivo- and IVM/TVF-derived, respectively) of hatched blastocysts were significantly higher (P < 0.01) than in the control groups (2.9 and 0%, in vivo and IVM/IVF-derived, respectively). These results suggested that dual culture conditions are required to optimize an in vitro culture system for the development of the porcine embryo in vitro.
Potassium (K(+)) concentration in the mammalian oviduct and uterus is particularly interesting due to its unusually high concentration (12 to 25 mM) compared with that in the blood stream (3 to 6 mM). In this study we examined the effects of various K(+) concentrations in the fertilization medium on polyspermy and subsequent in vitro development of porcine oocytes. In the absence of K(+) in the fertilization medium, sperm penetration was not observed. The incidence of polyspermy was significantly higher in the fertilization medium that contained 6 or 12 mM K(+) as compared with 3 mM K(+). The mean number of sperm penetrated in oocytes in medium with 6 and 12 mM K(+) was higher than in medium with 3 mM K(+). The addition of 3, 6 or 12 mM K(+) to the fertilization medium did not significantly affect the proportion of zygotes that developed to the blastocyst stage (14.1, 12.4 and 15.0%, respectively). Chlorotetracycline (CTC) analysis was used to determine the capacitation and acrosome reaction of spermatozoa incubated for 3 h with various concentrations of K(+). The number of acrosome reacted spermatozoa decreased with increasing K(+) concentration. These results suggest that extracellular K(+) in the fertilization medium affects sperm acrosome reaction which may be related to the sperm penetration.
We compared the developmental ability and gene expression of in vivo- and IVM/IVF-derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF-derived and 129 in vivo-collected zygotes was used to examine developmental ability and gene expression following DNA microinjection. When either DNA injected or noninjected zygotes were cultured for 4 d in NCSU 23 followed by 5 d in Eagle's minimal essential medium (EMEM), the percentages of zygotes developing to blastocysts and hatched blastocysts were higher (P < 0.05) compared with groups cultured in NCSU 23 alone. The percentages of injected embryos reaching the morula and blastocyst stages were significantly lower (P < 0.05) than that of noninjected control embryos whether in vivo or IVM/IVF derived. The percentages of morula and blastocyst stage embryos expressing the gene were higher in the in vivo-derived embryos than in IVM/IVF-derived embryos. A lower proportion of (67 to 77%) mosaicism was observed in the in vivo-derived embryos than in IVM/IVF (90 to 100%) derived embryos. The total cell number of blastocysts cultured in both NCSU 23 and EMEM media was significantly higher than that of blastocysts cultured continuously in NCSU 23. Our results suggest that this dual culture system enhanced embryo viability following microinjection of foreign DNA.
Baroreflex dysfunction (BRD) is an uncommon but perplexing clinical entity that occurs after an operation performed in the head and neck. Cases of BRD have occasionally been reported after bilateral carotid endarterectomies and in rare brain-stem tumors. We describe, for the first time to our knowledge, BRD in a patient after nasopharyngectomy and bilateral carotid isolation for recurrent nasopharyngeal carcinoma.