System Xc- Antiporter Inhibitors: Azo-Linked Amino-Naphthyl-Sulfonate Analogues of Sulfasalazine.

The cystine/glutamate antiporter system Xc- (SXc-) mediates the exchange of intracellular L-glutamate (L-Glu) with extracellular L-cystine (L-Cys2). Both the import of L-Cys2 and the export of L-Glu take on added significance in CNS cells, especially astrocytes. When the relative activity of SXc- overwhelms the regulatory capacity of the EAATs, the efflux of L-Glu through the antiporter can be significant enough to trigger excitotoxic pathology, as is thought to occur in glioblastoma. This has prompted considerable interest in the pharmacological specificity of SXc- and the development of inhibitors. The present study explores a series of analogues that are structurally related to sulfasalazine, a widely employed inhibitor of SXc-. We identify a number of novel aryl-substituted amino-naphthylsulfonate analogues that inhibit SXc- more potently than sulfasalazine. Interestingly, the inhibitors switch from a competitive to noncompetitive mechanism with increased length and lipophilic substitutions, a structure-activity relationship that was previously observed with aryl-substituted isoxazole. These results suggest that the two classes of inhibitors may interact with some of the same domains on the antiporter protein and that the substrate and inhibitor binding sites may be in close proximity to one another. Molecular modeling is used to explore this possibility.

High-resolution 3 T MRI of traumatic and degenerative triangular fibrocartilage complex (TFCC) abnormalities using Palmer and Outerbridge classifications.

AIM: To investigate the usefulness of high-resolution 3 T magnetic resonance imaging (MRI) for the evaluation of traumatic and degenerative triangular fibrocartilage complex (TFCC) abnormalities among three groups: patients presenting with wrist pain who were (a) younger than age 50 years or (b) age 50 or older (PT<50 and PT≥50, respectively), and (c) asymptomatic controls who were younger than age 50 years (AC). MATERIALS AND METHODS: High-resolution 3 T MRI was evaluated retrospectively in 96 patients, including 47 PT<50, 38 PT≥50, and 11 AC. Two board-certified radiologists reviewed the MRI images independently. MRI features of TFCC injury were analysed according to the Palmer classification, and cartilage degeneration around the TFCC was evaluated using the Outerbridge classification. Differences in MRI findings among these groups were detected using chi-square test. Cohen's kappa was calculated to assess interobserver and intra-observer reliability. RESULTS: The incidence of Palmer class 1A, 1C and 1D traumatic TFCC injury was significantly (p<0.05) higher in PT≥50 than in PT<50 (class 1A: 47.4% versus 27.7%, class 1C: 31.6% versus 12.8%, and class 1D: 21.1% versus 2.1%). Likewise, MRI findings of TFCC degeneration were observed more frequently in PT≥50 than in PT<50 (p<0.01). Outerbridge grade 2 or higher cartilage degeneration was significantly (p<0.01) more frequently seen in PT≥50 than in PT<50 (55.3% versus 17% in the lunate, 28.9% versus 4.3% in the triquetrum, 73.7% versus 12.8% in the ulna). CONCLUSION: High-resolution wrist MRI at 3 T enables detailed evaluation of TFCC traumatic injury and degenerative changes using the Palmer and Outerbridge classifications, with good or excellent interobserver and intra-observer reliability.

Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells.

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.

Therapeutic treatment of DMBA-induced mammary tumors with PPAR ligands.

The objective of this study was to evaluate the ability of troglitazone (a thiazolidinedione) and Wy-14,643 (a clofibrate) to inhibit progression of non-detectable and detectable mammary tumors in rats induced by 7,12 dimethylbenz(a)anthracene (DMBA) when compared to those receiving no treatment or tamoxifen. Although not as effective as tamoxifen in decreasing overall tumor incidence, Wy-14,643 reduced the percentage and number of malignant tumors that developed when compared to both troglitazone and control. Treatment of detectable tumors with either Wy-14,643 or troglitazone induced regression or stasis of total tumor volume in 40-50% of the animals, compared to only 10% in control and 65% in tamoxifen treated animals. Moreover, each PPAR ligand was as effective as tamoxifen in preventing additional tumor development. In summary, both PPAR ligands were more effective than no treatment in preventing tumor progression once detected. However, only the PPAR-alpha activator, Wy-14,643 was able to reduce the development of malignant tumors when administered prior to detection.

A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein.

As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbance, electron microscopy, fluorescent focus assay, and the "gold standard" plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10(-4)- to 10(-6)-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy + GFP + GAL). The two approaches yielded similar titers: 3.25 +/- 1.85 x 10(9) PFU/mL versus 3.46 +/- 0.76 x 10(9) green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7 x 10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.

Confocal imaging of flows in artificial venular bifurcations.

We describe a new experimental methodology for visualizing three-dimensional structures in microscopic tubes under flow conditions. Through the use of microfabrication techniques, artificial venular bifurcations are constructed from glass tubes with semicircular cross sections (radius = 50 mu). Aqueous fluorescent solutions are infused into the tubes at flow rates of about 1 microliter/min, a value comparable to blood flow in the microcirculation. The flow is imaged using a combination of confocal microscopy and three-dimensional image reconstruction software techniques. The quantitative accuracy of the experimental method is evaluated by measuring the "separation surface," a formation resulting from converging flows at a bifurcation. Details of the fabrication process, fluidics, confocal microscopy, image reconstructions, optical effects, and computations are described. We show the first three-dimensional visualization of a microscopic flow structure using confocal microscopy, and within certain limitations, quantitative agreement between the measured and computed positions of the separation surface.

A new method for blood velocimetry in the microcirculation.

We describe a simple new method for computing two-dimensional velocity vector fields of blood flows in microvascular networks. This method, known as optical flow, requires a time sequence of two or more images obtained by a process such as conventional videomicroscopy. There is no need to distinguish individual cells provided clear variations in the light intensity levels are present in the images. The result of the computation reveals the velocity distribution in the microvascular network and the geometry of the blood vessels. Although various implementations of the optical flow technique exist, we have found that the spatial correlation algorithm of Anandan performs best for microcirculatory flows. This report is an introduction to the application of optical flow to the microcirculation and provides instructions on how to obtain and run the codes.

Infant feeding decisions in the Missouri WIC Program.

A breastfeeding questionnaire was developed to help determine how we could better support our WIC mothers' efforts to breastfeed. The questionnaire was pilot tested in 12 local WIC agencies, wherein 878 participants returned the instrument for tabulation. Forty-six percent of the women who returned questionnaires initiated breastfeeding. The main support for women choosing to breastfeed came from their husbands. Women choosing not to breastfeed identified "self" as the person who most discouraged them from breastfeeding. Using this tool has helped to highlight the breastfeeding needs of our participants.

The teratogenic effects of valproic acid in human chondrogenesis in vitro.

The anticonvulsant drug valproic acid (VPA) is a known teratogen in humans. In general, anticonvulsants effect major systems in the embryo causing craniofacial, cardiovascular, neurological, urogenital, and major and minor skeletal defects. The limb defects associated with in utero VPA exposure include digital hypoplasia, ectrodactyly, radial ray aplasia, and proximal phocomelia. Human studies are limited to case reports and to retrospective and/or prospective studies. Although animal studies have demonstrated a teratogenic effect of VPA on skeletogenesis, these doses were well above the human therapeutic dose which makes extrapolation from these studies to humans difficult. The purpose of this research was to evaluate the potential deleterious effects of VPA on chondrogenesis, a process that occurs in human limb formation. To accomplish this goal, human chondrocytes were cultured in a three dimensional agarose gel and treated with VPA. The use of this model system was a novel approach to evaluate the teratogenic potential of VPA during chondrogenesis. The influence of VPA on human chondrocytes was monitored using histochemical, immunocytochemical, and morphological techniques. There was a decrease in mitotic activity and the extracellular matrix was modified. At human therapeutic doses, immunofluorescence revealed that type II collagen was reduced, while type I collagen increased. In addition, the alcian blue-staining matrices (i.e., sulfated proteoglycans) were reduced. Moreover, the Golgi apparatus had swelling in the trans-face cisternae suggesting that proteoglycan synthesis may be altered.

Caring for the medically indigent: who will pay the bill?

One of the most urgent problems facing society today is finding adequate resources to fund health care services for the indigent and medically indigent. In this article, the author describes the magnitude of the problem and suggests how trustees can participate in the search for a solution.

Ownership issues confront county hospitals.

Most often, a county decides to sell its hospital without considering all of the alternatives for revitalizing the hospital or the ultimate consequences of the sale. Issues and recommendations regarding sale to investor-owned chains and not-for-profit systems as well as other arrangements are examined in this article.

Financing health care in the 1980s.

An aging population, national health insurance, and the factors that drove up the cost of health care in the 1970s will accelerate cost increases in the 1980s.