Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

The structure-activity relationship study on 2-, 5-, and 6-position of the water soluble 1,4-dihydropyridine derivatives blocking N-type calcium channels

In order to find an injectable and selective N-type calcium channel blocker, we have performed the structure–activity relationship (SAR) study on the 2-, 5-, and 6-position of 1,4-dihydropyridine-3-carboxylate derivative APJ2708 (2), which is a derivative of Cilnidipine and has L/N-type calcium channel dual inhibitory activities. As a consequence of the optimization, 6-dimethylacetal derivative 7 was found to have an effective inhibitory activity against N-type calcium channels with more than 170-fold lower activity for L-type channel compared to that of APJ2708.     

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) drive protein translation in cells and hence these are essential enzymes across life. Inhibition of these enzymes can halt growth of an organism by stalling protein translation. Therefore, small molecule targeting of aaRS active sites is an attractive avenue from the perspective of developing anti-infectives. Febrifugine and its derivatives like halofuginone (HF) are known to inhibit prolyl-tRNA synthetase of malaria parasite Plasmodium falciparum. Here, we present functional and crystallographic data on P. falciparum prolyl-tRNA synthetase (PfPRS). Using immunofluorescence data, we show that PfPRS is exclusively resident in the parasite cytoplasm within asexual blood stage parasites. The inhibitor HF interacts strongly with PfPRS in a non-competitive binding mode in presence or absence of ATP analog. Intriguingly, the two monomers that constitute dimeric PfPRS display significantly different conformations in their active site regions. The structural analyses presented here provide a framework for development of febrifugine derivatives that can seed development of new anti-malarials.     

Evidence for pH dependent Zn2+influx in K562 erythroleukemia cells: studies using ZnAF-2F fluorescence and 65Zn2+ uptake

Using both ZnAF-2F (a Zn2+ specific fluorophore) and 65Zn2+, we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 microM), the initial rate of 65Zn2+ accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 microM for the Km and 0.203 nmol/mg/s for the Vmax were obtained for Zn2+ influx. 65Zn2+ uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pKa for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect.     

Kinetic analysis of the binding of guanine nucleotide to bovine brain smg p25A

Bovine brain smg p25A, a guanine nucleotide-binding protein with a Mr of about 25,000, bound specifically GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and GDP. The initial velocities of the binding of GTP gamma S to GDP-bound smg p25A and the dissociation of GDP from this protein increased by decreasing Mg2+ concentrations or increasing NaCl concentrations. The initial velocity of the binding of GTP gamma S to GDP-free smg p25A was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from smg p25A limits the binding of GTP to this protein, and suggest that there is a protein stimulating the dissociation of GDP from smg p25A and thereby stimulating the binding of GTP to this protein in mammalian tissues. In fact, the protein stimulating the dissociation of GDP, but not of GTP gamma S, from smg p25A was detected in bovine brain cytosol.     

Tissue-specific expression of a novel GTP-binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC-12 cells

We have purified a novel GTP-binding protein, designated as the smg-25A protein (smg p25A), from bovine brain membranes and determined its primary structure. In the present studies, the smg-25A mRNA levels in various tissues have been studied. The 1.6-kilobase smg-25A mRNA is detected in rat brain by Northern blot analysis. This mRNA is not detected in other rat tissues including thymus, lung, heart, liver, small intestine, kidney, and skeletal muscle. The 1.6-kilobase smg-25A mRNA is also detected in bovine adrenal medulla but not in the cortex. Moreover, this mRNA is detected in rat pheochromocytoma PC-12 cells and its level increases after differentiation of the cells into sympathetic neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP. This mRNA level does not increase in response to 12-O-tetradecanoylphorbol-13-acetate incapable of inducing differentiation. These results suggest that the smg-25A gene is specifically expressed in nerve tissues and that smg p25A plays a role in some neuronal functions.     

Restoring ovulation in beef donor cows with ovarian cysts by progesterone-releasing intravaginal silastic devices.

The objective of this study was to determine the efficacy of a progesterone-releasing intravaginal silastic device (Controlled Internal Drug Release: CIDR) for inducing ovulation in beef cows with persistent ovarian cysts. Fifteen cows with cysts and abnormal cycles for over 40 days were randomly assigned to receive either a single CIDR (CIDR group, n=9), or a CIDR containing no progesterone (blank CIDR) (BLANK group, n=6) for about 14 days. Determination of plasma progesterone levels at the beginning of CIDR treatment indicated 4 of 6 BLANK cows with non-luteinized cysts and 5 of 9 CIDR cows with non-luteinized cysts. In 5 of 6 BLANK cows, one follicular wave appeared and newly emerged dominant follicles increased in size up to 20 mm in diameter and persisted during the experiment, while one cow experienced estrus with spontaneous ovulation. In contrast, during CIDR treatment, 2 or 3 waves, in which dominant follicles were from 7 to 15 mm in diameter, appeared approximately at 7-day intervals. Within 3 days after CIDR removal, estrous behavior was detected followed by ovulation of the dominant follicle in the last wave. All CIDR cows resumed normal cyclicity with 2 follicular waves for over 2 months. Insertion of a CIDR caused a rapid increase of about 2 ng/mL in plasma progesterone. The levels were greater than 1.3 ng/mL until removal of a CIDR, then dropped under 0.3 ng/mL. Concentrations of plasma estradiol in BLANK cows increased during growth of the cystic follicles, with high levels greater than 10 pg/mL for over 10 days. In 4 of 5 cows with non-luteinized cysts, with high plasma estradiol on the day of CIDR insertion, CIDR treatment resulted in rapid decline of estradiol levels. During placement of the CIDR, estradiol levels showed no increase in the growth phase of a newly appeared dominant follicle. After CIDR removal, however, estradiol significantly increased associated with the growth of ovulatory follicles in all 9 cows. A transient increase in plasma FSH levels preceded detection of each follicular or cyst wave in both BLANK and CIDR cows. Pulse frequency and mean concentration of LH in cows with non-luteinized cysts showed values corresponding to those in normal follicular phase. However, throughout CIDR treatment, these parameters reduced to levels found in the normal luteal phase. In cows with luteinized cysts, parameters of LH secretion were as low as in the normal luteal phase before and during CIDR treatment, then increased significantly after CIDR removal. Present results indicate that treatment with CIDR proved effective in restoring ovulation and reestablishing normal cyclicity in beef donor cows with cysts persistent for a long period. The CIDR reduced and maintained LH secretion at normal luteal levels, thereby, inducing atresia of estrogen-active cysts and preventing formation of cysts from the newly emerged follicles.     

DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues

In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected. Gingival fibroblasts are the major constituents of gingival tissue. We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues [Biochem. Biophys. Res. Commun. 273 (2000) 1161]. To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis. We also studied the expression levels of these proteins by flow cytometric analysis (FACS). The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis. FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group. Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease. Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.     

Effects of pravastatin sodium on mevalonate metabolism in common marmosets

In experimental animals and humans, the concentration of serum mevalonate (MVA), a direct product of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is considered to reflect the activity of whole-body sterol synthesis. The relationship between the concentration of serum MVA and the activity of sterol synthesis in tissues, however, has not been fully clarified. In the present study, we examined MVA metabolism by using pravastatin, a liver-selective inhibitor of HMG-CoA reductase, and common marmosets, a good model animal for studying lipid metabolism. In the time course study, the maximal reduction in the concentration of serum MVA was observed 2 h after a single oral administration of 30 mg/kg pravastatin to common marmosets. We, therefore, examined the relationship between the concentrations of serum and hepatic MVA, and sterol synthesis in some tissues at this time point. Sterol synthesis was determined ex vivo in tissue slices by measuring the incorporation of [14C]acetate into digitonin-precipitable [14C]sterols. Pravastatin at 0.03-30 mg/kg reduced dose-dependently the activity of hepatic sterol synthesis, whereas no significant reduction of sterol synthesis was observed in other tissues such as intestine, kidney, testis and spleen, even with the highest dose (30 mg/kg). The liver-specific inhibition of sterol synthesis caused parallel reductions in the concentrations of both serum and liver MVA. In addition, there were good correlations between the concentration of either serum or hepatic MVA and the activity of hepatic sterol synthesis. These data indicate that the major origin of serum MVA is the liver, and that the concentration of serum MVA reflects the concentration of hepatic MVA and the activity of hepatic sterol synthesis 2 h after a single oral administration of pravastatin in common marmosets.