Effects of Ca2+ or Na+ on metallothionein expression in tilapia larvae (Oreochromis mossambicus) exposed to cadmium or copper.

The objectives of this study were to try to determine the reasons of the external Ca(2+) and Na(+) enhancement of Cd(2+) and Cu(2+) resistance in fish. Tilapia larvae at 3 days posthatch were exposed to (A) 0 (control), 40 microg/L Cd(2+), 40 microg/L Cd(2+) + 2 mM Ca(2+) (Cd/hyper-Ca), and 2 mM Ca(2+) or (B) 0 (control), 75 microg/L Cu(2+), 75 microg/L Cu(2+) + 0.52 mM Na(+) (Cu/hyper-Na), and 0.52 mM Na(+). After 48 hours, results indicated that (1) Cd/hyper-Ca and Cu/hyper-Na treatments showed decreased growth inhibition induced by the metals; (2) metal accumulation in Cd/hyper-Ca-treated larvae was lower compared with those exposed only to Cd; and (3) metallothionein (MT) expression was significantly higher in Cu/hyper-Na-treated larvae than in the group treated with Cu only. Taking all of this into account, either supplementary Ca(2+) or Na(+) in ambient water may help fish to maintain Ca(2+) or Na(+) homeostasis, which could decrease metal accumulation and its detrimental effects. Consequently, the fish increase MT expression and retard the growth inhibition caused by metals.

A single transcutaneous injection with Botox for dysthyroid lid retraction.

PURPOSE: To evaluate the safety and efficacy of injections with botulinum toxin type A (BTTA,Botox), given transcutaneously, in the treatment of upper lid retraction associated with thyroid eye disease (TED). METHODS: A total of 15 patients (21 eyes) with a stable (TED) condition, and a euthyroid state, were enrolled into the study. There were 12 females and three males from ages 23 to 52 years. A single injection, at the centrally superior tarsal border transcutaneously, aiming at the levator aponeurosis and Müller muscle, was administered into each eyelid with 5-6 U of Botox. All patients were followed regularly for 4-6 months. Any complications, such as ptosis, diplopia, pain,or lid ecchymosis were recorded. RESULTS: All patients, except one, experienced much reduction of palpebral fissure. The mean difference of MRD1 between pre- and postinjections of Botox at the first week was -3.1 mm, and the effect remained, at least, for 2 months. There were temporary complications of ptosis in three patients and vertical diplopia in two patients, lasting 3-4 weeks. CONCLUSIONS: A single transcutaneous injection with Botox for the treatment of thyroid lid retraction is safe and effective. Some minor complications may occur, such as ptosis and diplopia; however, it may offer an alternative and temporary method for patients with dysthyroid lid retraction, who are waiting for a staged operation of either an orbital decompression or a strabismus surgery or both.

Effects of inverse ratio ventilation versus positive end-expiratory pressure on gas exchange and gastric intramucosal PCO(2) and pH under constant mean airway pressure in acute respiratory distress syndrome.

BACKGROUND: In patients with acute respiratory distress syndrome, whether inverse ratio ventilation differs from high positive end-expiratory pressure (PEEP) for gas exchange under a similar mean airway pressure has not been adequately examined. The authors used arterial oxygenation, gastric intramucosal partial pressure of carbon dioxide (PiCO(2)), and pH (pHi) to assess whether pressure-controlled inverse ratio ventilation (PC-IRV) offers more benefits than pressure-controlled ventilation (PCV) with PEEP. METHODS: Seventeen acute respiratory distress syndrome patients were enrolled and underwent mechanical ventilation with a PCV inspiratory-to-expiratory ratio of 1:2, followed by PC-IRV 1:1 initially. Then, they were randomly assigned to receive PC-IRV 2:1, then 4:1 or 4:1, and then 2:1, alternately. The baseline setting of PCV 1:2 was repeated between the settings of PC-IRV 2:1 and 4:1. Mean airway pressure and tidal volume were kept constant by adjusting the levels of peak inspiratory pressure and applied PEEP. In each ventilatory mode, hemodynamics, pulmonary mechanics, arterial and mixed venous blood gas analysis, PiCO(2), and pHi were measured after a 1-h period of stabilization. RESULTS: With a constant mean airway pressure, PC-IRV 2:1 and 4:1 decreased arterial and mixed venous oxygenation as compared with baseline PCV 1:2. Neither the global oxygenation indices with oxygen delivery and uptake nor PiCO(2) and pHi were improved by PC-IRV. During PC-IRV, applied PEEP was lower, and auto-PEEP was higher. CONCLUSION: When substituting inverse ratio ventilation for applied PEEP to keep mean airway pressure constant, PC-IRV does not contribute more to better gas exchange and gastric intramucosal PiCO(2) and pHi than does PCV 1:2 for acute respiratory distress syndrome patients, regardless of the inspiratory-to-expiratory ratios.

Synthesis, characterization and preclinical formulation of a dual-action phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities.

In a systematic effort to develop a microbicide contraceptive capable of preventing transmission of human immunodeficiency virus (HIV), as well as providing fertility control, we have previously identified novel phenyl phosphate derivatives of zidovudine (ZDV) with 5-halo 6-alkoxy substitutions in the thymine ring and halo substitution in the phenyl moiety respectively. Here, we describe the synthesis, characterization, and successful preclinical formulation of our lead compound, 5-bromo-6-methoxy-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), which exhibits potent anti-HIV and sperm immobilizing activities. The anti-HIV activity of WHI-07 was tested by measuring viral p24 antigen production and reverse transcriptase activity as markers of viral replication in HIV-1 infected human peripheral blood mononuclear cells (PBMC). WHI-07 inhibited replication of HIV in a concentration-dependent fashion with nanomolar IC50 values. The effects of WHI-07 on human sperm motion kinematics were analysed by computer-assisted sperm analysis (CASA), and its effects on sperm membrane integrity were examined by confocal laser scanning microscopy (CLSM), and high-resolution low-voltage scanning electron microscopy (HR-LVSEM). WHI-07 caused cessation of sperm motility in a concentration- and time-dependent fashion. The in-vitro cytotoxicities of WHI-07 and nonoxynol-9 (N-9) were compared using normal human ectocervical and endocervical epithelial cells by the MTT cell viability assay. Unlike N-9, WHI-07 had no effect upon sperm plasma and acrosomal membrane integrity. N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, whereas WHI-07 was selectively spermicidal. The in-vivo vaginal absorption and vaginal toxicity of 2% gel-microemulsion of WHI-07 was studied in the rabbit model. The sperm immobilizing activity of WHI-07 was 18-fold more potent than that of N-9. Over a 10 day period, there was no irritation or local toxicity to the vaginal epithelia or systemic absorption of WHI-07. Therefore, as a potent anti-HIV agent with spermicidal activity, and lack of mucosal toxicity, WHI-07 may have the clinical potential to become the active ingredient of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

Synthesis of dual function (5R,6R)- and (5S,6S)-5-bromo-6-methoxy-5,6-dihydro-AZT-5'-(para-bromophenyl methoxyalaninyl phosphate) as novel spermicidal and anti-HIV agents.

We synthesized a novel compound, 5-bromo-6-methoxy-5,6-dihydro-AZT-5'- (p-bromophenyl methoxyalaninyl phosphate), which had an EC50 value of 5 microM in sperm motility assays. This is > 1 log10 better than that of the detergent spermicide nonoxynol-9 (EC50 81 microM). The compound also displayed a potent anti-human immunodeficiency virus (HIV) activity with an IC50 value of 0.005 microM in HIV replication assays, which was virtually identical to that of AZT (IC50 0.006 microM) and > 2 log10 more potent than that of nonoxynol-9 (IC50 2.2 microM). The promising results reported herein recommend the further development of the dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives as a new class of vaginal contraceptives capable of preventing the sexual transmission of HIV while providing fertility control for women who are at high risk of acquiring HIV by heterosexual transmission. These dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives may also have utility in curbing domestic and wildlife animal retroviral transmissions.

AZT-5'-(p-bromophenyl methoxyalaninyl phosphate) as a potent and non-toxic anti-human immunodeficiency virus agent.

The potency and selectivity index of the AZT-phenyl phosphate derivatives in thymidine kinase (TK)-deficient T cells were substantially enhanced by introducing a single para-bromo substitutent in the phenyl moiety. AZT-5'-(p-bromophenyl methoxyalaninyl phosphate) was 43-fold more potent than AZT-5'-(phenyl methoxyalaninyl phosphate) and was fivefold more potent than AZT in inhibiting human immunodeficiency virus (HIV) replication in TK-deficient CEM cells.

Aryl phosphate derivatives of bromo-methoxy-azidothymidine are dual-function spermicides with potent anti-human immunodeficiency virus.

Detergent-based vaginal microbicides, in addition to their high contraceptive failure rates, cause mucosal erosion and local inflammation that might increase the risk of heterosexual human immunodeficiency virus (HIV) transmission. In a systematic effort to identify a microbicide contraceptive potentially capable of preventing the sexual transmission of HIV as well as providing fertility control, a series of novel aryl phosphate derivatives of 5-bromo-6-methoxy-3'-azido-3'-deoxythymidine (AZT; zidovudine) were synthesized and examined for dual anti-HIV and sperm-immobilizing activity (SIA). Whereas AZT displayed potent anti-HIV activity (IC50 = 0.006 microM) but lacked SIA (EC50 > 300 microM), two 5-bromo-6-methoxy-aryl phosphate derivatives of AZT, compounds WHI-05 and WHI-07, exhibited potent anti-HIV activity as well as SIA. The IC50 (HIV) and EC50 (SIA) values for WHI-07 were 439-fold and 13.5-fold lower, respectively, than those for the detergent-based virucidal spermicide, nonoxynol-9 (N-9). Sperm motion kinematics using computer-assisted sperm motion analysis combined with confocal laser scanning microscopy, high-resolution low-voltage scanning, and transmission electron microscopy demonstrated that both WHI-05 and WHI-07 cause a complete and irreversible loss of sperm motility in a concentration- and time-dependent fashion without concomitantly affecting the sperm acrosomal membrane integrity. In experiments designed to assess the fertilizing capacity of treated sperm, preincubation of sperm with either compound resulted in a concentration-dependent loss of the ability to adhere to and penetrate zona-free hamster eggs as well as inhibition of binding to human zona. WHI-07 applied intravaginally prior to artificial insemination of epididymal sperm drastically reduced fertility in hormonally primed CD-1 mice. Unlike the intravaginal application of N-9, repetitive intravaginal application of WHI-07 did not damage the vaginal epithelium or cause local inflammation. Structure-function relationship analyses showed that the addition of bromo-methoxy functional groups to AZT was essential for, and the aryl phosphate derivatization contributory to, the SIA of both compounds. Compounds WHI-05 and WHI-07 may be useful as dual-function vaginal contraceptives for women who are at high risk for acquiring HIV/acquired immunodeficiency virus syndrome by heterosexual vaginal transmission.

Diffusion-dependent kinetic properties of glyoxalase I and estimates of the steady-state concentrations of glyoxalase-pathway intermediates in glycolyzing erythrocytes.

The diffusion-dependent kinetic properties of the yeast glyoxalase I reaction have been measured by means of viscosometric methods. For the glyoxalase-I-catalyzed isomerization of glutathione (GSH)-methylglyoxal thiohemiacetal to S-D-lactoylglutathione, the k(cat)/Km (3.5 x 10(6) M(-1) s(-1), pH 7, 25 degrees C) undergoes a progressive decrease in magnitude with increasing solution viscosity, using sucrose as a viscogenic agent. The viscosity effect is unlikely to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, as the magnitude of k(cat)/Km for the slow substrate GSH-t-butylglyoxal thiohemiacetal (3.5 x 10(3) M(-1) s(-1), pH 7, 25 degrees C) is independent of solution viscosity. Quantitative treatment of the data by means of the Stokes-Einstein diffusion law suggests that catalysis will be about 50% diffusion limited under conditions where [substrate] << Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. In a related study, the steady-state concentrations of glyoxalase-pathway intermediates in glycolyzing human erythrocytes are estimated to be in the nanomolar concentration range, on the basis of published values for the activities of glyoxalase I and glyoxalase II in lysed erythrocytes and the steady-state rate of formation of D-lactate in intact erythrocytes. This is consistent with a model of the glyoxalase pathway in which the enzyme-catalyzed steps are significantly diffusion limited under physiological conditions.

Development of the lateral eye of American horseshoe crabs: visual field and dioptric array.

We used a precision two-circle goniometer mounted to the stage of a compound microscope to determine the optical alignment and to measure the entrance aperture diameter of individual cuticular cones in the dioptric array of the lateral eye of juvenile horseshoe crabs in order to learn about the development of the visual field. Our results show that the extent of the visual field of juvenile horseshoe crabs with prosomal lengths about 20% of adult size (14-21 mm) is about 70% that of the visual field of adult horseshoe crabs (prosomal lengths: 100+ mm). The visual field of such juvenile animals covers between 77 and 85 deg vertically and 140 and 145 deg horizontally. Assuming that the dioptric array is uniform and square packed, the average interommatidial angle of the juvenile animals is between 5.6 and 6.0 deg as compared to 4.6 deg for an adult animal. The diameter of the entrance aperture of individual cuticular cones increases markedly with increasing animal size. In addition, we noted a statistically significant trend for entrance aperture diameters to increase from anterior to posterior within the eye for animals of all sizes. There may be a slight trend for entrance aperture diameters to increase from dorsal to ventral within the eye. Our results indicate that the extent of the visual field and the resolution of the lateral eye approach adult values in advance of animals' reaching sexual maturity.

Bovine liver formaldehyde dehydrogenase. Kinetic and molecular properties.

The dimeric formaldehyde dehydrogenase from bovine liver has been resolved into three nearly homogeneous enzyme forms by the successive use of ion-exchange, affinity, and ampholine (chromatofocusing) chromatography. The different enzyme species were isolated in the approximate proportions 3:2:1, having pI values of 6.5, 6.2, and 6.0, respectively. The subunit molecular weights of the three forms are all similar (Mr congruent to 41,000), on the basis of sodium dodecyl sulfategel electrophoresis. The enzyme species appear to arise from covalent differences unrelated either to partial proteolysis during isolation or to differential sialization of homodimeric protein. Human liver contains a single major form and two minor forms of formaldehyde dehydrogenase having pI values very similar to those found for the bovine liver enzyme. The macroscopic kinetic constants (V, V/K) for the three forms of the dehydrogenase from bovine liver are all similar in magnitude, using NADH and S-hydroxymethylglutathione as substrates. The isotope-sensitive hydride transfer step is not significantly rate-limiting during catalysis by any of the forms, as evidenced by the near-unity primary deuterium isotope effects on both V and V/KS (for S-hydroxymethylglutathione); catalysis may be limited by the rate of dissociation of at least one (and possibly both) of the product molecules. In support of rate-limiting dissociation of NAD+ in the normal reaction, V increases by approximately 22-fold and isotope effects of approximately 1.4 are observed on both V and V/KS, using the coenzyme analog 3-acetylpyridine adenine dinucleotide. Product dissociation from the active site appears to be accelerated by the presence of dilute denaturing agents, perhaps indicative of a rate-limiting conformational transition associated with product release.

Stereoselective inhibition of human placental aromatase.

We have synthesized the (19R)- and (19S)-isomers (2 and 3 respectively) of 10 beta-oxiranylestr-4-ene-3,17-dione. The configurations and conformations of these compounds were established by X-ray crystallographic analysis. Each of these compounds is a powerful competitive inhibitor of human placental microsomal aromatase, and stereoselectivity of inhibition was observed (Ki values for 2 and 3 were 7 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of oxirane oxygen to the heme iron of the enzyme. The (19R)- and (19S)-10 beta-thiiranes (6 and 7) corresponding to 2 and 3 have been synthesized from the oxiranes by a stereospecific process. The thiiranes are very effective competitive inhibitors of placental aromatase, and show even greater stereoselectivity in binding than the oxiranes (Ki values for 6 and 7 were 1 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of thiirane sulfur to heme iron.