The physicochemical parameters of marker compounds and vehicles for use in in vitro percutaneous absorption studies

In order to prepare for a validation study to compare percutaneous absorption through reconstructed human epidermis with ex vivo skin absorption through human and animal skin, nine test compounds, covering a wide range of physicochemical properties were selected, namely: benzoic acid; caffeine; clotrimazole; digoxin; flufenamic acid; ivermectin; mannitol; nicotine; and testosterone. The donor and receptor media for the test substances, the addition of a solubiliser for the lipophilic compounds, as well as the stability and solubility of the test substances in the vehicles, were systematically analysed. Hydrophilic molecules, being freely soluble in water, were applied in buffered saline solutions. In order to overcome solubility restrictions for lipophilic compounds, the non-ionic surfactant, Igepal CA-630, was added to the donor vehicle, and, in the case of clotrimazole and ivermectin, also to the receptor fluid. The model molecules showed a suitable solubility and stability in the selected donor and receptor media throughout the whole duration of the test.     

Gas exchanges and yield responses of mungbean (Vigna radiata L. Wilczek) genotypes differing in flooding tolerance

Flooding-induced changes in leaf gas exchanges, grain yield, and yield-related parameters of mungbean were evaluated employing two flood-tolerant (GK48 and VC3945A) and one flood-susceptible (Vo1982A-G) genotypes. Three flooding regimes viz. 1, 3 and 7-day were imposed at vegetative, flowering, and pod-fill stages. Flooding caused a drastic reduction in photosynthesis rates (P _n), irrespective of flooding duration. However, the flooded plants recovered P _n to a large extent depending on genotypes. Used genotypes showed a significant variation in P _n during and after flooding. Post-flooding recovery in P _n of GK48 and VC3945A was more pronounced at the vegetative and flowering stages than the pod-fill stage. At the pod-fill stage, only plants of GK48 survived when flooding prolonged for 7 days. Flooded plants showed higher intercellular CO₂ concentrations (C _i), and reduced stomatal conductance (g _s). However, during recovery, P _n increased significantly along with reduced C _i in flood-tolerant GK48 and VC3945A genotypes. In contrast, C _i remained high and P _n recovery was minimal in flood-susceptible Vo1982A-G genotype. This implies that mesophyll tolerance rather than stomatal factor might be the major limitation of P _n recovery in a susceptible genotype. Very weak relationship between P _n and transpiration rate (T _r) indicated low water use efficiency (WUE) in flooded plants, but subsequent recovery of both the parameters, suggesting higher WUE, particularly in tolerant genotypes. Seed yield of mungbean was the product of number of pods per plant and seed size, and longer the flooding period, the lower were the pods per plant at the flowering and pod-fill stage. Flooding reduced seed yield in all the three genotypes, but the extent of reduction was much less in flood-tolerant GK48 and VC3945A. Higher yield of flood-tolerant genotypes may be attributed to the rapid recovery of leaf gas exchanges.     

Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by Rhodococcus sp. Strain DN22

A unique metabolite with a molecular mass of 119 Da (C₂H₅N₃O₃) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Molecular characterization of <Emphasis Type="Italic">AIPL1</Emphasis> gene region in the Iranian population: application of novel informative haplotypes and detection of mutational founder effect

Leber’s congenital amaurosis (LCA) is considered as one of the main causes of congenital blindness. In view of the genetically heterogeneous nature of the disease, indirect diagnosis using linkage analysis has proven to be useful in molecular diagnosis procedure. Mutations in AIPL1 gene are one of the leading causes of LCA. In the present study, the application of three single nucleotide polymorphic (SNP) markers related to the gene, including rs7212734, rs11658369 and rs8066853 was evaluated for the first time in the Iranian population. The markers were genotyped using tetra-primer ARMS PCR in 154 unrelated healthy individuals. Haplotype frequency and other characteristics of the markers were examined by using the GENEPOP, PowerMarker and Cubic Exact Solution software. The data indicated the presence of six different haplotypes in the Iranian population. Among them, three haplotypes showed high informativeness with frequencies ≥0.05. Three unrelated Iranian LCA families were analyzed. The p.W278* mutation in exon 6 of AIPL1 was found in all the families using APEX microarray chips. SNP analysis for the families showed the conservation of T?A?A haplotype linked to p.W278* mutation. All families bearing the mutation were from the Isfahan province and a founder effect was suggested in this population. Prenatal diagnosis using the markers led to the successful prediction of the fetus genotypes in all the at risk pregnancies and showed that rs7212734, rs11658369 and rs8066853 can be considered as the three informative markers for linkage analysis in carrier detection and molecular diagnosis of LCA in the Iranian population.     

Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ER alpha cells: bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridization

We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178-1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1-2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2-3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro.     

Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine and Its Mononitroso Derivative Hexahydro-1-Nitroso-3,5-Dinitro-1,3,5-Triazine by Klebsiella pneumoniae Strain SCZ-1 Isolated from an Anaerobic Sludge

In previous work, we found that an anaerobic sludge efficiently degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), but the role of isolates in the degradation process was unknown. Recently, we isolated a facultatively anaerobic bacterium, identified as Klebsiella pneumoniae strain SCZ-1, using MIDI and the 16S rRNA method from this sludge and employed it to degrade RDX. Strain SCZ-1 degraded RDX to formaldehyde (HCHO), methanol (CH₃OH) (12% of total C), carbon dioxide (CO₂) (72% of total C), and nitrous oxide (N₂O) (60% of total N) through intermediary formation of methylenedinitramine (O₂NNHCH₂NHNO₂). Likewise, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) was degraded to HCHO, CH₃OH, and N₂O (16.5%) with a removal rate (0.39 μmol·h⁻¹·g [dry weight] of cells⁻¹) similar to that of RDX (0.41 μmol·h⁻¹·g [dry weight] of cells⁻¹) (biomass, 0.91 g [dry weight] of cells·liter⁻¹). These findings suggested the possible involvement of a common initial reaction, possibly denitration, followed by ring cleavage and decomposition in water. The trace amounts of MNX detected during RDX degradation and the trace amounts of hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine detected during MNX degradation suggested that another minor degradation pathway was also present that reduced —NO₂ groups to the corresponding —NO groups.     

Interaction of daunomycin antibiotic with histone H(1): ultraviolet spectroscopy and equilibrium dialysis studies

Using ultraviolet spectroscopy and equilibrium dialysis techniques, we have investigated the interaction of anticancer drug, daunomycin with calf thymus histone H(1) chromosomal protein in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA at room temperature. The UV spectroscopy results show that daunomycin (5.0-100 microM) decreases the absorbance of histone H(1) at 210-230 nm and induces hypochromicity in the absorption spectrum of the protein. The equilibrium dialysis data show that daunomycin binds to histone H(1) and the binding process is positive cooperative with two binding sites as Scatchard plot and Hill coefficient confirm it. The results suggest that daunomycin binds to histone H(1) and changes its conformation.     

Characterization of different reactive lysines in bovine heart mitochondrial porin

Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channel's selectivity and its voltage dependence.     

Clathrin-mediated endocytosis of Na+,K+-ATPase in response to parathyroid hormone requires ERK-dependent phosphorylation of Ser-11 within the alpha1-subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.     

Formation of three new flavones by differentiating callus cultures of andrographis paniculata

Differentiating tissue cultures of Andrographis paniculata produce three new flavones, 5-hydroxy-7,8,2′-trimethoxy-, 5,2′-dihydroxy-7,8-dimethoxy- and 5-hydroxy-7,8-dimethoxy-flavones. Flavones are not synthesized by the de-differentiated callus. Closely related flavones have been isolated from intact plants of Andrographis species.