Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7µM), verapamil (0.6 vs. 0.7µM) and prazosin (0.099 vs. 0.033µM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

Microbiological profile of culture-proven cases of exogenous and endogenous endophthalmitis: a 10-year retrospective study.

PURPOSE: To identify the microbial aetiology of infectious endophthalmitis and to determine the in vitro antibacterial susceptibilities of bacterial isolates. METHODS: A retrospective analysis was carried out of all patients presenting between January 1997 and December 2006 with clinically diagnosed infectious endophthalmitis who underwent microbiological evaluation. Intraocular specimens (aqueous and vitreous fluids) were collected from all cases of clinically suspected infectious endophthalmitis. In addition to intraocular aspirates, blood specimens from endogenous endophthalmitis, and corneal and scleral scrapes from relevant cases were also collected. The collected intraocular specimens, blood specimens, and corneal and scleral scrapes were subjected to microbiological evaluation. RESULTS: Samples from 955 patients with endophthalmitis underwent microbiological analysis, of which 424 (44.4%) were found to be culture positive. Of 424, 364 (85.8%) had bacterial growth and the remaining 60 (14.2%) had fungal growth. Among post-surgical endophthalmitis, Gram-negative bacilli (75%) were found to be the predominant cause for developing fulminant onset, Staphylococcus spp. (68.6%) for acute, and Streptococcus spp. (75%) for chronic onset of infections, whereas in post-traumatic endophthalmitis, Gram-negative bacilli (65.2%) were found to be the predominant cause for fulminant onset, Gram-positive bacillus (28.4%) for acute onset, and fungi (52.3%) for chronic onset of infections. Endophthalmitis associated with microbial keratitis was mainly caused by filamentous fungi (37.2%) and Gram-negative bacilli (37.2%). Overall, gatifloxacin (97.7%) showed highest activity against bacterial isolates followed by ciprofloxacin (95.9%) and ofloxacin (95.1%). CONCLUSION: Gram-negative bacilli cause predominantly fulminant onset, Staphylococci and Gram-positive bacilli acute, and Streptococci, Nocardia, and fungi chronic endophthalmitis. Gatifloxacin demonstrated greatest efficacy against these bacterial isolates.

Identification of the type II Na(+)-Pi cotransporter (Npt2) in the osteoclast and the skeletal phenotype of Npt2-/- mice.

We previously reported that a type II sodium phosphate (Na(+)-Pi) cotransporter (Npt2) protein is expressed in osteoclasts and that Pi limitation decreases osteoclast-mediated bone resorption in vitro. We also demonstrated that mice homozygous for the disrupted Npt2 gene (Npt2-/-) exhibit a unique age-dependent bone phenotype that is associated with significant hypophosphatemia. In the present study, we sought to identify the Npt2 cDNA in mouse osteoclasts and characterize the impact of Npt2 gene ablation on osteoclast function and bone histomorphometry. We demonstrate that the osteoclast Npt2 cDNA sequence is identical to that of the proximal renal tubule and, thus, not an isoform or splice variant thereof. Histomorphometric analysis revealed that, at 25 days of age, Npt2-/- mice exhibited a reduction in osteoclast number and eroded perimeters, relative to wild-type mice. Moreover, although the number of metaphyseal trabeculae was reduced in 25-day-old Npt2-/- mice, trabecular bone volume was normal due to increased trabecular width. At 115 days of age, the decrease in osteoclast index persisted in Npt2-/- mice relative to wild-type littermates. However, mineralizing and osteoblast surfaces and bone formation rates were increased, and, although trabecular number was still reduced, trabecular bone volume was higher than that of wild-type mice. These data demonstrate a link between osteoclast activity and trabecular development in young Npt2-/- mice, and suggest that an age-related adaptation to Npt2 deficiency is apparent in osteoclast and osteoblast function and bone formation.

Regulation of neuronal thyroid hormone receptor alpha 1 mRNA by hydrocortisone, thyroid hormone and retinoic acid.

The regulation of thyroid hormone receptor alpha 1 (TR alpha 1) mRNA by hydrocortisone (HC), thyroid hormone (T3) and retinoic acid (RA) has been studied in mixed and neuronal primary cultures of cells dissociated from fetal rat cerebra. Steady-state levels of TR alpha 1 mRNA were increased as much as 5-fold at 13 days of development by 0.3 microM HC in both mixed and neuronal-enriched cultures. The regulation of TR alpha 1 mRNA by HC appears to be mainly limited to neurons. This conclusion is based on two observations. First, stimulation by HC occurs in cultures highly enriched in neurons at approximately the same time and extent as that seen in mixed-cell cultures (containing neurons, oligodendroglia and astroglia). Second, the stimulation reaches a peak at 13 days in mixed-cell cultures, an age at which neurons but not glia differentiate. In most cases, neither T3 (20 nM) nor RA (10(-7) M) stimulated an increase in TR alpha 1 mRNA steady-state levels. The exception was that RA increased TR alpha 1 mRNA levels at 13 days in culture, but at no other stage of development. In both types of cultures, RA and T3 separately and together produced as much as a complete inhibition of the stimulation by HC. Regulation by T3, when it occurred, was always negative. The fact that T3 can strongly repress the induction of TR alpha 1 mRNA by HC demonstrates that T3 can function through negative cooperativity as a potent regulator of its own receptor in brain.

In vitro induction of androgenic haploids in Safflower (Carthamus tinctorius L.).

The influence of culture medium on induction of androgenic calli was examined with five different basal media. MS medium was the most responsive in inducing callus. Differences in induction of calli among ten genotypes revealed that the most responsive genotype was a local cultivar, Mangira, with 48.6% anthers initiating callus formation. The influence of temperature pre-treatment (5°±1°C) for varying periods (0 to 15 days) on immature capitula prior to inoculation of anthers on the medium revealed that the percentage of anthers inducing callus increased till 3-5 days of pre-treatment. The effect of physiological conditions of anther donor plants grown in the field and in green houses on induction and re-differentiation have shown that the field grown anther donor plants exhibited optimum response. Shoot regeneration was observed on MS supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) and rhizogenesis on MS (half-strength) medium, supplemented with NAA (0.1 mg/l) and 1% sucrose. Cytological studies of anther derived plants showed two ploidy levels, where the haploids were predominant (64%).

Shear velocity of muscle tissues.

Investigations on shear wave velocities in a large number of elastoplastic muscle tissues of dead animals (ox, sheep, and goat) were carried out at 1 MHz frequency by the ultrasonic pulse transmission technique and their rigidity and stiffness modulus estimated after determining the density due to displacement in mercury. Information on compressional wave velocity and acoustic impedance, together with the elastic properties and some results on shear velocity anisotropy are also presented. It is interesting to note that irrespective of organ, species and direction of measurement, the results of velocity, rigidity and stiffness fall within very narrow limits owing to their nearly identical amino acid composition.