Structural diversities of active site in clinical azole-bound forms between sterol 14alpha-demethylases (CYP51s) from human and Mycobacterium tuberculosis

To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET-bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single gx and gz values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.     

rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

Some Physicochemical Properties of Invertase of Candida utilis

This study examined the microbial transformation of carbazole (CZ) by an isolated bacterium that can use CZ as a sole carbon and nitrogen source. The strain identified as Pseudomonas stutzeri produced a large amount of anthranilic acid (AA) from CZ in the medium containing a nonionic surfactant. In dialysis culture using ion-exchange resin, 7.9 g/liter (58mm) of AA was accumulated from 15g/liter (90mm) of CZ and the molar yield of AA reached about 64%.     

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( γ-GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of γ-GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

In theXenopus oocyte heterologous expressionsystem, the electrophysiological characteristics of rabbit ClC-2current and its contribution to volume regulation were examined.Expressed currents on oocytes were recorded with a two-electrodevoltage-clamp technique. Oocyte volume was assessed by taking picturesof oocytes with a magnification of ×40. Rabbit ClC-2 currentsexhibited inward rectification and had a halide anion permeabilitysequence of Cl  Br  I  F. ClC-2 currents wereinhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid(9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similarto those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of ratClC-2. During a 30-min perfusion with hyposmolar solution, currentamplitude at 160 mV and oocyte diameter were compared amongthree groups: oocytes injected with distilled water, oocytes injectedwith ClC-2 cRNA, and oocytes injected with ClC-2NT cRNA (an openchannel mutant with NH₂-terminaltruncation). Maximum inward current was largest in ClC-2NT-injectedoocytes (5.9 ± 0.4 µA), followed by ClC-2-injected oocytes(4.3 ± 0.6 µA), and smallest in water-injected oocytes(0.2 ± 0.2 µA), whereas the order of increase in oocytediameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2NT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling wassmallest in oocytes with the largest expressed currents suggest thatClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to ahyposmolar environment.