Effect of sulfite treatment on total antioxidant capacity, total oxidant status, lipid hydroperoxide, and total free sulfydryl groups contents in normal and sulfite oxidase-deficient rat plasma

Sulfites, which are commonly used as preservatives, are continuously formed in the body during the metabolism of sulfur-containing amino acids. Sulfite oxidase (SOX) is an essential enzyme in the pathway of the oxidative degradation of sulfite to sulfate protecting cells from sulfite toxicity. This article investigated the effect of sulfite on total antioxidant capacity (TAC), total oxidant status, lipid hydroperoxide (LOOH), and total free sulfydryl groups (-SH) levels in normal and SOX-deficient male albino rat plasma. For this purpose, rats were divided into four groups: control, sulfite-treated, SOX-deficient, and sulfite-treated SOX-deficient groups. SOX deficiency was established by feeding rats a low molybdenum diet and adding to their drinking water 200 ppm tungsten. Sulfite (70 mg/kg) was administered to the animals via their drinking water. SOX deficiency together with sulfite treatment caused a significant increase in the plasma LOOH and total oxidant status levels. -SH content of rat plasma significantly decreased by both sulfite treatment and SOX deficiency compared to the control. There was also a significant decrease in plasma TAC level by sulfite treatment. In conclusion, sulfite treatment affects the antioxidant/oxidant balance of the plasma cells of the rats toward oxidants in SOX-deficient groups.     

A filamentous phage display system for N-linked glycoproteins

We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X₁-N-X₂-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 × 10⁵ per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the −2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype–phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.     

Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 Å above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp⁹⁸. Furthermore, the 7-position of the imipramine ring is found vicinal to Phe³³⁵, and the pocket lined by Ala¹⁷³ and Thr⁴³⁹ is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).     

Joint analysis of the DRD5 marker concludes association with attention-deficit/hyperactivity disorder confined to the predominantly inattentive and combined subtypes

Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable, heterogeneous disorder of early onset, consisting of a triad of symptoms: inattention, hyperactivity, and impulsivity. The disorder has a significant genetic component, and theories of etiology include abnormalities in the dopaminergic system, with DRD4, DAT1, SNAP25, and DRD5 being implicated as major susceptibility genes. An initial report of association between ADHD and the common 148-bp allele of a microsatellite marker located 18.5 kb from the DRD5 gene has been followed by several studies showing nonsignificant trends toward association with the same allele. To establish the postulated association of the (CA)(n) repeat with ADHD, we collected genotypic information from 14 independent samples of probands and their parents, analyzed them individually and, in the absence of heterogeneity, analyzed them as a joint sample. The joint analysis showed association with the DRD5 locus (P=.00005; odds ratio 1.24; 95% confidence interval 1.12-1.38). This association appears to be confined to the predominantly inattentive and combined clinical subtypes.     

Complications after reconstruction by plate and soft-tissue free flap in composite mandibular defects and secondary salvage reconstruction with osteocutaneous flap

Reconstruction of composite defects of the mandible is a challenging problem. Although the use of an osteocutaneous free flap, alone or in combination with another soft-tissue free flap, is generally accepted to be optimal, the bony reconstruction is sometimes undervalued, especially when the cancer is advanced. In such situations, reconstruction is often performed with a reconstruction plate covered with a soft-tissue free flap. Between January of 1997 and July of 2000, 80 patients with composite or extensive composite oromandibular defects underwent treatment with a reconstruction plate and a soft-tissue free flap. All of the patients were male, and the ages of the patients at the time of treatment ranged from 32 to 78 years (mean, 51 years). Tumors were classified as stage IV in 56 patients (70 percent), whereas the remaining 24 patients (30 percent) had recurrent carcinomas. The titanium mandibular reconstruction system manufactured by Stryker (Freiburg, Germany) was used to bridge the mandibular defects. The soft-tissue free flaps used for wound and plate coverage were as follows: anterolateral thigh flap (n = 75), radial forearm flap (n = 3), transverse rectus abdominis myocutaneous flap (n = 1), and tensor fasciae latae flap (n = 1). Five patients with recurrent carcinomas and 10 with stage IV carcinomas (18.75 percent) died 2 to 6 months after the operation and were excluded from the study. The remaining 65 patients were monitored for an average follow-up period of 22 months (range, 6 to 40 months). During that period, one or more complications occurred for 45 patients (69.2 percent). Plate exposure was the most common complication and was observed for 30 patients (46.15 percent). Twenty of the 65 patients (30.8 percent) required secondary salvage reconstruction with a fibula osteoseptocutaneous flap. The decision to perform a secondary salvage procedure was based on the general health of the patient, the extent of local disease, and the severity of the complications. Patients underwent salvage operations after an average of 11.5 months (range, 6 to 26 months). The major reasons for the second operation were as follows: reconstruction plate exposure (n = 12), soft-tissue deficiency and mandibular contour deformation of the lateral face (n = 7), intraoral contracture and lack of a gingivobuccal sulcus (n = 6), trismus (n = 4), and osteoradionecrosis of the mandible (n = 2). The total flap survival rate was 90 percent (18 of 20 free flaps). In two cases, the skin paddles of the fibula osteoseptocutaneous flaps exhibited partial failure and were revised with pedicled pectoralis major and deltopectoral flaps. The reconstruction plate and free soft-tissue flap procedure for the reconstruction of composite defects of the oromandibular region has many late complications, which eventually necessitate reconstruction of the mandible with an osteocutaneous free flap.     

Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa

The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).     

Effects of the nasal valve on acoustic rhinometry measurements: a model study.

The influence of nasal valve on acoustic rhinometry (AR) measurements was evaluated by using simple nasal cavity models. Each model consisted of a cylindrical pipe with an insert simulating the nasal valve. The AR-determined cross-sectional areas beyond the insert were consistently underestimated, and the corresponding area-distance curves showed pronounced oscillations. The area underestimation was more pronounced in models with inserts of small passage area. The experimental results are discussed in terms of theoretically calculated "sound-power reflection coefficients" for the pipe models. The reason for area underestimation is reflection of most of the incident sound power from the barrier at the front junction between the pipe and the insert. It was also demonstrated that the oscillations are due to low-frequency acoustic resonances in the portion of the pipe beyond the insert. The results suggest that AR does not provide reliable information about the cross-sectional areas of the nasal cavity posterior to a significant constriction, such as pathologies narrowing the nasal valve area. When the passage area of the nasal valve is decreased, the role of AR as a diagnostic tool for the entire nasal cavity becomes limited.     

Effects of paranasal sinus ostia and volume on acoustic rhinometry measurements: a model study.

We used pipe models to investigate the effects of paranasal sinus ostium size and paranasal sinus volume on the area-distance curves derived by acoustic rhinometry (AR). Each model had a Helmholtz resonator or a short neck as a side branch that simulated the paranasal sinus and sinus ostium. The AR-derived cross-sectional areas posterior to the ostium were significantly overestimated. Sinus volume affected the AR measurements only when the sinus was connected via a relatively large ostium. The experimental area-distance curve posterior to the side branch showed pronounced oscillations in association with low-frequency acoustic resonances in this distal part of the pipe. The experimental results are discussed in terms of theoretically calculated "sound-power reflection coefficients" for the pipe models used. The results indicate that the effects of paranasal sinuses and low-frequency acoustic resonances in the posterior part of the nasal cavity are not accounted for in the current AR algorithms. AR does not provide reliable information about sinus ostium size, sinus volume, or cross-sectional area in the distal parts of nasal cavity.