Autoimmune ovarian disease: mechanism of disease induction and prevention

Investigation of the versatile models for autoimmunity of the ovary and other selected organs has contributed to our understanding of the following aspects of autoimmunity: the mechanism of T cell molecular mimicry; T-->B epitope spreading, as a basis for autoantibody diversification, and as a link between organ-specific and systemic autoimmunity; the localization of genetic loci potentially influencing multiple autoimmune diseases; and the elucidation of regulatory T cells as a component of physiological self tolerance.     

N-cadherin-catenin interaction: necessary component of cardiac cell compartmentalization during early vertebrate heart development

During early heart development the expression pattern of N-cadherin, a calcium-dependent cell adhesion molecule, suggests its involvement in morphoregulation and the stabilization of cardiomyocyte differentiation. N-cadherin's adhesive activity is dependent upon its interaction with the intracellular catenins. An association with alpha-catenin and beta-catenin also is believed to be involved in cell signaling. This study details the expression patterns of alpha-catenin, beta-catenin, and gamma-catenin, during definition of the cardiac cell population as distinct compartments in the anterior regions of the chick embryo between stages 5 and 9. The restriction of N-cadherin/catenin localization at stage 5+ from a uniform pattern in vivo, to specific cell clusters that demarcate areas where mesoderm separation is initiated, suggests that the N-cadherin/catenin complex is involved in boundary formation and in the subsequent cell sorting. The latter two processes lead to the specification and formation of the somatic and cardiac splanchnic mesoderm. N-cadherin colocalized with alpha- and beta-catenin at the cell membrane before and during the time that its expression becomes restricted to the lateral mesoderm and continues cephalocaudad into stage 8. These proteins continue to colocalize in the myocardium of the tubular heart. Plakoglobin is not expressed in this region during stages 6-8, but is detected in the myocardium later at stage 13. The observed in vivo expression patterns of alpha-catenin, beta-catenin, and plakoglobin suggest that these proteins are directly linked with the developmental regulation of cell junctions, as cardiac cells become stably committed and phenotypically differentiated to eventually form a mature myocardium. The localization of N-CAM also was analyzed during these stages to determine whether the N-cadherin-catenin localization was unique or whether other cell adhesion molecules were expressed similarly. The results indicate that the unique pattern of N-cadherin expression is not shared with N-CAM. We also show that perturbation of N-cadherin using a function perturbing N-cadherin antibody (NCD-2) inhibits normal early heart development and myogenesis in a cephalocaudad, stage-dependent manner. We propose a model whereby myocardial cell compartmentalization also defines the endocardial population. The presence of beta-catenin suggests that a similar signaling pathway involving Wnt (wingless)-mediated events may function in myocardial cell compartmentalization during early vertebrate heart development, as in Drosophila contractile vessel development.     

Lymph node involvement and tumor depth in rectal cancers: an analysis of 805 patients

BACKGROUND: Superficial rectal tumors are said to involve regional lymph nodes rarely. This presumption must be proven beyond any doubt if less radical surgery is to be offered for such patients. PATIENTS AND METHODS: Eight hundred five cases (467 males; median age, 64 (range, 19-97) years) of rectal cancer were reviewed. RESULTS: Lymph node positivity, number of lymph nodes involved, lymphatic vessel, and venous and perineural invasion were significantly increased with increasing depth of invasion of tumor through the bowel wall in univariate analysis. The percentage of lymph node involvement at each tumor depth was as follows: T1, 5.7 percent; T2, 19.6 percent; T3, 65.7 percent; T4, 78.8 percent. Overall lymph node involvement was 59 percent. For patients younger than 45 years of age, the percentage of lymph node involvement was 33.3, 30, 69.3, and 83.3 percent compared with 3.1, 8.4, 64.2, and 78.8 percent for patients aged 45 years or above for T1, T2, T3, and T4, respectively. CONCLUSION: Increased depths of tumor penetration beyond T1 and age less than 45 years have an excessive incidence of lymph node positivity. The finding of lymphatic vessel invasion on biopsy is highly indicative of lymph node metastasis.     

Mass loss in urethane/TEGDMA- and Bis-GMA/TEGDMA-based resin composites during post-cure heating

OBJECTIVES: This research examined weight loss of commercial UDMA/TEGDMA- and Bis-GMA/TEGDMA-based resin composites at a variety of post-cure temperatures. Weight loss profiles of individual monomer components were also tested at elevated temperature: Bis-GMA, ethoxylated Bis-GMA (EBis-GMA), urethane dimethacrylate (UDMA), and triethyleneglycol dimethacrylate (TEGDMA). METHODS: Disc-shaped composite specimens (1 x 5 mm, approximately 50 mg) were light-cured and then isothermally post-cure heated in a thermogravimetric analysis (TGA) unit at either 50 degrees, 75 degrees, 100 degrees, 125 degrees or 150 degrees C. A single specimen was made for each post-cure temperature for each product (a total of 10 discs). Individual monomer components were heated to 800 degrees C. Filler and organic phase weight percentages were determined by ashing cured composite in the TGA. Weight loss differences between resin systems at various post-cure temperatures were analyzed using linear regression. RESULTS: For each type of composite, loss of volatile component increased with both elevated post-cure temperature as well as duration of heat exposure. Using recommended post-cure temperature and time (125 degrees C for 7.5 minutes), there was no difference in weight loss profile between the two products: both exhibited 1.3% loss of resin component. After 10 min of heating, the Bis-GMA-based product always demonstrated a greater weight loss than the UDMA material. Weight loss could not be attributed to any specific monomer. SIGNIFICANCE: Specimen weight loss during post-cure heating may result in a depletion of leachable, unreacted material at the restoration surface, possibly enhancing material properties at that location. This decrease would also potentially reduce the biological impact of leachable materials. Loss of volatile components from post-cure heating would affect the accuracy of infrared spectroscopic techniques in determining monomer conversion values.     

In vivo observations and electron microscopy in the treatment of experimental HSV keratitis with monoclonal antibodies

In vivo observations and electron microscopy showed that topical use of anti-HSV monoclonal glycoprotein antibodies produced marked antiviral effects in inhibiting the development of experimental herpetic keratitis in rabbits and in protecting the susceptible corneal cells. As a new biological product, the anti-HSV monoclonal antibodies may provide a new approach to the treatment of HSV keratitis.     

HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium

The E6 and E7 genes of HPV-16 or HPV-18 both are necessary for effective immortalization of primary human genital keratinocytes. To analyse the individual role of E6 and E7 genes in dysregulating cell growth, we cloned the HPV-16 E6, E7 and E6/E7 genes into retroviruses. Primary human keratinocytes (PHK) were then infected with these retroviruses and selected in differentiation-inducing medium (high calcium and serum). The E6/E7 retroviruses were the most effective at inducing differentiation-resistant colonies. Intermediate numbers of colonies were induced by E6 and low numbers by E7. Interestingly, only cultures infected with E7 and E6/E7 retroviruses showed a significant proportion of cells progressing into the S phase, consistent with our earlier studies showing that E7 is required for the efficient immortalization of genital keratinocytes. Accompanying this entry into S phase, the E7 or E6/E7 transduced cells expressed high levels of cyclins A, B and E, but lower levels of cyclin D. In addition, cdc-2, cdk-2 and cdk-4 were also increased. No significant differences were detected in the expression of c-myc and c-fos between the vector and any of the transduced cells. Keratinocytes infected with the E7 retrovirus exhibited decreased levels of Rb protein and increased levels of p53, whereas cells infected with E6-expressing retroviruses displayed normal levels of Rb protein and decreased levels of p53. Finally, E7 induced a three-fold increase in bcl-2 expression. Our results indicate that the HPV-16 E7 gene alone is sufficient to bypass keratinoctye growth arrest induced by serum and calcium exposure and that the discordant expression of several cell regulatory proteins accompanies this unregulated proliferation.     

Electrophysiological properties of cultured neonatal rat dorsal horn neurons containing GABA and met-enkephalin-like immunoreactivity

We have developed a culture of neurons dissociated from the most superficial laminae of the neonatal rat spinal cord dorsal horn (DH). By using the perforated patch-clamp technique, we distinguished four types of neurons based on their firing properties in response to intracellular injection of 900 ms lasting current pulses. Type 1 neurons were characterized by a tonic firing. Type 2 neurons displayed marked spike accommodation and fired brief (<500 ms) bursts of action potentials, whereas type 3 neurons fired a single spike. Type 4 neurons exhibited different types of firing patterns, but all of them possessed a time-dependent inwardly rectifying current activated by membrane hyperpolarization. Met-enkephalin-like immunoreactivity (met-ENK-LI) and glutamic acid decarboxylase-like immunoreactivity (GAD-LI) were colocalized in 42% of the neurons (n = 59), which were previously identified electrophysiologically. Type 1-4 neurons represented respectively 4, 64, 20, and 12% of the population of neurons colocalizing met-ENK-LI and GAD-LI. We conclude that the electrophysiological properties of DH neurons present in our cultures are similar to those described in acute slice or hemisected spinal cord preparations and that met-ENK-LI and GABA-LI are preferentially colocalized in type 2 neurons.     

Detection of protein A produced by Staphylococcus aureus with a fiber-optic-based biosensor

Staphylococcus aureus is a pathogen important in causing human infections and intoxication. A sensitive fiber-optic that produces evanescent waves was developed for the detection of protein A, a product secreted only by S. aureus. In the immunosensor, a 40-mV argon-ion laser that generated laser light at 488 nm was used together with plastic optical fiber and antibodies to protein A were physically adsorbed onto the fiber. The principle of the detection involved a sandwich immunoassay with fluorescein isothiocyanate conjugated with anti-(protein A) immunoglobulin G to produce signals of the antigen-antibody reaction. The detection limit was 1 ng of protein A per milliliter. The fiber-optic immunosensor could be used for rapid and specific detection of S. aureus in clinical specimens and foods.     

Cellular thiols as a determinant of responsiveness to menadione in cardiomyocytes

The role of intracellular thiols in menadione-mediated toxicity was studied in neonatal rat cardiomyocytes. The sensitivity of cardiomyocytes to menadione was greater than that of skeletal muscle cells and 3T3 fibroblasts. Before cell degeneration, menadione induced marked depletion of intracellular thiols and an increase of oxidized glutathione. The sensitivity of these cells to menadione correlated with the level of depletion of intracellular thiols. After incubation of cardiomyocytes with menadione, glutathione reductase activity was inhibited and lipid peroxidation was increased. Both dicumarol (an inhibitor of DT-diaphorase) and diethyldithiocarbamate (an inhibitor of superoxide dismutase) enhanced the capacity of menadione to induce cellular damage and to cause depletion of intracellular glutathione. Decreasing intracellular glutathione by pretreatment of cells with N-ethylmaleimide or buthionine sulphoximine also increased menadione-induced cell degeneration. Preincubation with cysteine or dithiothreitol suppressed the capacity of menadione to damage the cells. Menadione-induced lipid peroxidation was also suppressed by the same treatment. These results show that the oxidative stress induced by menadione in cardiomyocytes results in the depletion of glutathione and protein thiols. Both DT-diaphorase and superoxide dismutase can protect cells from the toxicity of menadione. Cellular thiols are determinants of the responsiveness to menadione.     

Direct fractionation of proteins in particle-containing feedstocks by a filter paper pieces-based DEAE-cellulose column chromatography. Rapid, robust and low-cost capturing procedure for protein

Goldfish were classically conditioned to a mixture of two pulse trains differing in both repetition rate and the spectral profile of the pulses. Animals were then tested for generalization to single pulse trains having one or the other spectral profile presented at a variety of repetition rates. Generalization functions of repetition rate were qualitatively similar to those obtained following conditioning to either of the pulse trains alone. Thus, the spectral profile of each pulse type was appropriately associated with the repetition rate at which that pulse type was presented during conditioning. These results indicate that the two concurrent pulse trains making up the conditioning stimuli were analyzed independently, forming two auditory streams. When either of the two pulse trains were presented with a 500 ms onset asynchrony, stream segregation was enhanced. These and other results suggest that many fundamental features of the human sense of hearing are widely shared among vertebrate animals, and may have developed first among fishes.