Changes in the intracellular accumulation and distribution of metallothionein in rat liver and kidney during postnatal development

Metallothionein (MT) bound to zinc and copper was detected in high concentration in fetal and newborn rat livers by a cadmium saturation method. The levels of both hepatic zinc and MT remained high for the first 14 days after birth and decreased to adult levels by 24 days of age. There was a direct linear relationship between hepatic metallothionein and zinc concentrations during the first 31 days after birth. The ratio of MT to zinc levels also decreased with age suggesting a rapid degradation of MT during postnatal development. Immunohistochemical localization of MT by peroxidase-antiperoxidase technique, using a specific antibody to MT, showed intense intranuclear staining for MT in fetal and newborn rat liver which persisted until Day 9. The nuclear MT staining decreased with age; at 11 days it was equal both in nucleus and cytoplasm and at 14 days, MT was localized mainly in the cytoplasm, similar to adult rat liver pattern. The intranuclear localization of MT in neonates could be considered as a typical fetal-neonatal morphological pattern and its subsequent presence in the cytoplasm, an adult pattern.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Optimization of orally bioavailable alkyl amine renin inhibitors

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC₅₀ of 0.83 nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10 mg/kg resulted in >20 h reduction of blood pressure in a double transgenic rat model of hypertension.     

Biphenyl/diphenyl ether renin inhibitors: filling the S1 pocket of renin via the S3 pocket

Structure-based design led to the discovery of a novel class of renin inhibitors in which an unprecedented phenyl ring filling the S1 site is attached to the phenyl ring filling the S3 pocket. Optimization for several parameters including potency in the presence of human plasma, selectivity against CYP3A4 inhibition and improved rat oral bioavailability led to the identification of 8d which demonstrated antihypertensive efficacy in a transgenic rat model of human hypertension.     

The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development

Nucleotide excision repair in Arabidopsis thaliana differs from other eukaryotes as it contains two paralogous copies of the corresponding XPB/RAD25 gene. In this work, the functional characterization of one copy, AtXPB1, is presented. The plant gene was able to partially complement the UV sensitivity of a yeast rad25 mutant strain, thus confirming its involvement in nucleotide excision repair. The biological role of AtXPB1 protein in A. thaliana was further ascertained by obtaining a homozygous mutant plant containing the AtXPB1 genomic sequence interrupted by a T-DNA insertion. The 3' end of the mutant gene is disrupted, generating the expression of a truncated mRNA molecule. Despite the normal morphology, the mutant plants presented developmental delay, lower seed viability and a loss of germination synchrony. These plants also manifested increased sensitivity to continuous exposure to the alkylating agent MMS, thus suggesting inefficient DNA damage removal. These results indicate that, although the duplication seems to be recent, the features described for the mutant plant imply some functional or timing expression divergence between the paralogous AtXPB genes. The AtXPB1 protein function in nucleotide excision repair is probably required for the removal of lesions during seed storage, germination and early plant development.     

Spirocyclic ureas: Orally bioavailable 11β-HSD1 inhibitors identified by computer-aided drug design

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11β-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.     

Discovery and optimization of adamantyl carbamate inhibitors of 11β-HSD1

Synthesis of 2-adamantyl carbamate derivatives of piperidines and pyrrolidines led to the discovery of 9a with an IC₅₀ of 15.2 nM against human 11β-HSD1 in adipocytes. Optimization for increased adipocyte potency, metabolic stability and selectivity afforded 11k and 11l, both of which were >25% orally bioavailable in rat.     

Cloning, characterization and site-directed mutagenesis of canine renin

Inhibition of renin has been shown to be successful in managing hypertension and maintaining cardiac health. Canine models have played a key role in preclinical assessment of renin inhibitors. Here we report the cloning of canine prorenin gene. The amino acid sequence of mature canine renin was approximately 70% identical to that of human renin. The full-length prorenin was expressed in HEK 293 cells, purified and converted to its active form by trypsin-mediated cleavage of the 43 residue propeptide. The mature enzyme was characterized by steady-state kinetics using a peptide corresponding to the canine angiotensinogen sequence, Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser-OH (cleavage between Leu(10)-Leu(11)). The reaction followed Michaelis-Menten kinetics with a K(M) of 120 microM and a second-order rate constant (k(cat)/K(M)) of 1.7 x 10(5) M(-)(1)s(-)(1). The enzyme was inhibited by various human renin inhibitors, but at reduced potency compared to the human renin. The basis of the species specificity was investigated by mutagenesis. Based on primary sequence and structural alignments, three mutants were prepared (G149S-S150T, V286L, G149S-S150T-V286L). Each mutant yielded catalytically active enzymes with lower specific activities than native canine renin. V286L had the greatest effect on substrate specificity, while G149S, S150T mutations produced enzymes with inhibitor profiles similar to human renin.     

Interaction between pesticides and essential metal copper increases the accumulation of copper in the kidneys of rats

Male Sprague-Dawley rats, weighing 175-200 g, six per group were fed AIN 93M diet (CON) or diets containing 500 mg Ca (LCa), 7 mg Zn (LZn), 2 mg Cu (LCu), 60 mg Zn (HZn), or 12 mg Cu (HCu) per kilogram of diet in the following combinations: control (CON), LCa+LZn (LC+Z), LCa+LZn+LCu (LC+Z+C), or HZn+HCu (HZ+C) without or with a pesticides mixture (PM); Endosulfan, Thiram, and Acephate were added to the diets at 25% of LD50/kg. Rats were fed for 2 wk (small intestinal changes) or 4 wk (tissues changes). Plasma Zn was 47% lower than CON in the experimental groups. Plasma Cu and ceruloplasmin concomitantly decreased in the LC+Z+C group and increased with the addition of PM. Kidney Cu was 40% lower in LC+Z+C group, than CON and increased by 31% with PM; in the HZ+C+PM group, kidney Cu was 38% higher than the HZ+C group. Mucosal and small intestines Ca declined by 47% in all experimental groups; PM increased Zn in the LC+Z+C and HZ+C groups; PM further decreased intestinal and mucosal Cu retention in the LC+Z+C and HZ+C groups. Data suggest that low levels of PM in the diet can induce Cu accumulation in the kidney when dietary Zn and Cu are low or high.     

Design and optimization of renin inhibitors: Orally bioavailable alkyl amines

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC₅₀ of 0.47 nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.