Increased expression of prion protein is associated with changes in dopamine metabolism and MAO activity in PC12 cells

Prion diseases of humans and animals occur following infection with infectious agents containing PrP(Sc) or in situations in which there is a mutation of the prion protein (PrP) gene. The cellular prion protein (PrP(C)) is a sialoglycoprotein that is expressed predominantly in neurons. PrP(C) is converted into a pathogenic form of PrP (PrP(Sc)), which is distinguishable from PrP(C) by its relative resistance to protease digestion. A number of postulates have been advanced for the function of normal PrP (PrP(C)), but this issue has not been resolved. To investigate the function(s) of PrP(C), we established clonal PC12 cell lines, which have elevated PrP(C) expression. The results show that there were alterations in dopamine metabolism and in monoamine oxidase (MAO) activity in transfected PC12 cells that overexpress PrP(C). There was an increase in concentration of DOPAC, a metabolite of dopamine, and in MAO activity in cells overexpressing PrP(C). MAO is involved in oxidative degradation of dopamine (DA). Our data suggest that PrP(C) plays a role in DA metabolism by regulating MAO activity.     

Progression of burn wound depth by systemical application of a vasoconstrictor: an experimental study with a new rabbit model

The final depth of a necrosis resulting from burn trauma is determined within 3 days. The zone of stasis has the potential for complete regeneration or there may be ischemic influences that lead to necrosis. In our model, we examined the dermal influence of vasoconstrictors with reference to the development of burn necrosis. On the backs of New Zealand white rabbits (4.0–4.5 kg) standardized lesions were made with a heated aluminum stamp at 80°C, 14 s in duration.

The lesions were intradermal, whereby the border zone of the coagulated tissue was found in the middle two quarters of the dermis in 100% of untreated animals after 72 h. For dermal vasoconstriction epinephrine in a dose of 0.5 μg/kg/min was used.

There were two groups of seven animals each. One group received epinephrine and the dosage was dependent on the clinical state of the animal. Several cycles were administered within a 3-day period. The reduction of skin perfusion was documented by Laser–Doppler-flowmetry. After 3 days, the skin with the lesions was excised and using a hematoxylin dye, a histological examination followed. The parameter used to determine the efficacy was the thickness of the uncoagulated part of the excised dermis.

Over a period of 48 h, an average of 2.3 epinephrine cycles of average of 88 min per animal in duration resulted in an average reduction of skin diffusion of 41%. The uncoagulated part of the dermis in the epinephrine group was 28.6% average; in the control group, this was 43.5%. The statistical analysis revealed significant differences with a p-value of 0.0312 (significant, when value is less than 0.05). The test results indicate that temporary reduction of skin perfusion through external administration of vasocontrictors may lead to progression of burn necrosis in our animal model.

Clinically, this result indicates that for patients with burn injuries and systemic inflammatory response syndrome who have insufficient volume therapy, the administration of vasocontrictors may produce similar results in the injured area.     

Facilitators and barriers to use of the female condom: qualitative interviews with women of diverse ethnicity

Women in the United States, particularly African-Americans and Hispanics, are at increased risk for HIV. The female condom now offers women a potentially important option for HIV prevention, yet few efforts have been made to increase its use. To elucidate strategies to promote the use of the female condom, we conducted in-depth interviews with 62 women recruited from the four major racial/ethnic groups of the U.S. (African-American, Asian-American, Hispanic, and white). Subject recruitment took place at a family planning clinic in San Francisco during 1996-97. We identified four major types of facilitators and barriers to use of the female condom: mechanical, psychosexual, interpersonal, and situational. Specifically, the mechanical facilitators and barriers included positive and negative aspects of the device, and difficulty with insertion. The psychosexual factors were female empowerment, more options for contraception and disease prevention, discomfort with vaginal insertion, and condom use norms. The interpersonal factors included: enhanced communication, relationship status, partner preferences, and partner objections. Finally, the situations that made women disinclined to use the device were: no access to the female condom when having sex and using other forms of contraceptives. The implications of these findings for HIV prevention and future research are discussed.     

Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells

PURPOSE: The loss of corneal endothelial cells associated with aging and possibly other causes has been speculated to be related to exposure to reactive oxygen species (ROS). The current study was conducted to investigate, by use of photosensitizers, the underlying mechanisms involved in the death of bovine corneal endothelial cells (BCENs) caused by ROS. METHODS: BCEN cells in primary culture were treated with a photosensitizer (riboflavin or rose bengal) with light exposure. The patterns of cell damage and death were assessed using an acridine orange-ethidium bromide differential staining method, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. Antioxidants, including catalase, L-histidine, salicylic acid, and superoxide dismutase, were used to determine the types of ROS involved. Activation of nuclear factor (NF)-kappaB was examined by fluorescent immunocytochemistry with anti-p65 antibody. RESULTS: Light-irradiated riboflavin or rose bengal resulted in a significant decrease in viability of BCEN cells. Chromosomal condensation and fragmentation were observed in apoptotic cells, and membrane lysis and damage of cell ultrastructures were observed in necrotic cells. Riboflavin induced apoptosis at 30 minutes and thereafter and induced necrosis after 2 hours. Rose bengal was shown to cause similar effects within half the time required for the effects of riboflavin. Catalase and salicylic acid were found to provide protection for BCENs from cytotoxic effects of riboflavin, and L-histidine was found to protect BCENs from cytotoxicity induced by rose bengal. Kinetic studies using immunocytochemistry showed that NF-kappaB was translocated into the nucleus within 15 minutes and 30 minutes after treatment with rose bengal and riboflavin, respectively. CONCLUSIONS: The cytotoxic effects of photo-irradiated riboflavin and rose bengal are shown to be mediated by two distinct but parallel pathways, one leading to apoptosis and the other to necrosis. Possible involvement of NF-kappaB in cell death is suggested. These findings provide potential leads for future investigation into the molecular mechanisms of loss of corneal endothelial cells related to aging, oxidative stress, and possibly other similar causes.     

Primary orbital Ewing sarcoma in a middle-aged woman

A 43-year-old woman had unilateral exophthalmos caused by primary orbital Ewing sarcoma. Specialized immunohistochemical stains, primarily MIC-2 (CD99), aided in the diagnosis of Ewing sarcoma. Twenty-two months after radiotherapy and multiagent chemotherapy, the patient remained tumor free. To our knowledge, this is the first reported case of orbital Ewing sarcoma to present in an adult beyond the fourth decade of life.     

Effect of topically applied 0.1% dexamethasone on endothelial healing and aqueous composition during the repair process of rabbit corneal alkali wounds

PURPOSE: The effects of topical dexamethasone on the endothelial healing and the change of aqueous compositions were investigated during the repair process of alkali-wounded rabbit cornea. METHODS: A central corneal alkali wound was produced by a 60 sec application of a 5.5 mm round filter paper soaked in 1N NaOH onto one eye of each rabbit. The eyes subsequently were treated topically with either 0.1% dexamethasone or a balanced salt solution (BSS) 4 times per day for 8 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The concentrations of ascorbic acid, glucose, and the ions, Na+, K+, Ca2+ and Mg2+, were measured in the aqueous humor. RESULTS: Endothelial healing in control (alkali-wounded but not treated with dexamethasone) corneas showed a biphasic pattern of healing: an initial short-term healing for the first week and then a late long-term healing following the secondary endothelial breakdown. Topical administration of 0.1% dexamethasone deterred endothelial healing during the early period and prevented the secondary endothelial breakdown. However, the total repair process of endothelium was accelerated by the dexamethasone-treatment. Among the various components of the aqueous humor examined, ascorbic acid seemed the most sensitive to change caused by the alkali injury and dexamethasone treatment. CONCLUSIONS: The present data indicate that dexamethasone may have a therapeutic potential in the management of endothelial healing after corneal alkali injury.     

Identification of urinary metabolites of clemastine after oral administration to man

The metabolism of clemastine, 2-[2-[1-(4-chlorophenyl)-1-phenylethoxylethyl])-1-methylpyrrolidin e, has been studied in three adult male volunteers after a single oral dose of 20 mg as the fumarate. After enzymatic hydrolysis solvent extracts of urine were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide-ammonium iodide and analysed by gas chromatography-mass spectrometry. The structures of metabolites were determined on the basis of electron impact and chemical ionization mass spectra and the identities of some (e.g. carbinol, 4-chlorobenzophenone and 4-chlorophenylstyrene) were confirmed by use of authentic standards. The principal route of metabolism of clemastine in man involves direct oxidation, O-dealkylation (fission of the ether bond), aromatic hydroxylation, aliphatic oxidation, alcoholic dehydration, and then enzymatic hydrolysis. Of the total amount of metabolites excreted in the urine 35% was carbinol (metabolite M3, major metabolite), 15% was M1, 17% was M2, 11% was M4, 9% was M5, 8% was M6 and 5% was M7.     

Cadmium inhibits albumin endocytosis in opossum kidney epithelial cells

Chronic exposure to cadmium results in proteinuria. To gain insights into the mechanism by which cadmium inhibits the protein transport in the renal proximal tubule, we investigated the effects of cadmium on the receptor-mediated endocytosis of albumin, using fluorescein isothiocyanate-labeled bovine serum albumin (FITC-albumin) as a model substrate and opossum kidney cell line (OK cell) as a proximal tubular cell model. Cell monolayers grown to confluence were treated with 100 microM CdCl(2) for 60 min at 37 degrees C, washed, and tested for FITC-albumin uptake (37 degrees C) and surface binding (4 degrees C). The amounts of FITC-albumin uptake and binding were quantified by fluorimetrically determining the cell-adherent fluorescence. Both the binding and uptake of FITC-albumin by OK cells appeared to be saturable and inhibitable by unlabeled albumin in the medium, indicating that specific receptor sites were involved. The uptake of FITC-albumin was inhibited by agents that interfere with the formation of endocytotic vesicle (hypertonic mannitol), endosomal acidification (NH(4)Cl), and vesicular trafficking (cytochalasin D and nocodazole), confirming that the uptake occurred via the process of receptor-mediated endocytosis. In cells treated with cadmium, the specific FITC-albumin uptake was significantly attenuated, and this was due to a reduction in V(max) and a rise in K(m). These changes in kinetic parameters were similar to those induced by NH(4)Cl. The binding of FITC-albumin to the apical surface of OK cells was inhibited by cadmium treatment, and this was attributed to a reduction in B(max). The values of K(d) and its pH dependency were not altered by cadmium treatment. The formation of endocytotic vesicles, as judged by fluid phase endocytosis of FITC-inulin, was not changed by cadmium treatment. These results indicate that the receptor-mediated endocytosis of albumin is impaired in cadmium-treated OK cells most likely due to a defect in endosomal acidification and the attendant fall in ligand-receptor dissociation, which impairs receptor recycling and the overall efficiency of endocytosis.