A continuous striatal infusion of 6-hydroxydopamine produces a terminal axotomy and delayed behavioral effects

Rat models of Parkinson's disease typically employ a rapid nigral injection of 6-hydroxydopamine (6-OHDA) to produce a near-complete loss of nigrostriatal dopamine neurons, and thus model end stage disease. The present report describes the use of a continuous, low dose infusion of 6-OHDA into the striatum which produces a terminal axotomy of nigrostriatal dopamine neurons and protracted behavioral response. A solution of 6-OHDA in 0.4% ascorbate, delivered at 37°C from osmotic minipumps, was stable for 8 days as determined by its retained toxicity to a dopaminergic neuroblastoma cell line. The continuous infusion of 0.2 μg 6-OHDA per h did not affect the striatal uptake of [³H]GABA, [³H]choline, or [³H]glutamate but reduced [³H]dopamine uptake by 55% within 1.5 days after the start of the infusion. The striatal infusion of 6-OHDA produced a dose-dependent reduction of striatal dopamine and DOPAC levels but did not alter HVA, 5-HT, or 5-HIAA. An increase in amphetamine-induced ipsiversive rotations occurred within 1.5 days after the acute striatal injection of 20 μg or 30 μg of 6-OHDA but required 4 days to develop with the continuous 6-OHDA infusion. The topography of the lesion mapped by [³H]mazindol binding showed that, begining by 1.5 days, a diffuse depletion of terminals encompassed much of the striatum in the 30 μg acute injection group, whereas in the continuously infused rats, the lesion was apparent only by 4 days and was restricted to a smaller and more completely lesioned area. Unlike acutely lesioned animals, continuously infused rats revealed no obvious loss of dopamine neurons in the pars compacta by 5 weeks after 6-OHDA. The continuous striatal infusion of 6-OHDA can produce a topographically limited terminal axotomy of dopamine neurons and a protracted behavioral impairment.     

Mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides.

Highly acidic compounds that are difficult to ionize by matrix-assisted laser desorption ionization give excellent spectra when mixed with a basic peptide or protein to form a noncovalent complex. This phenomenon makes it possible to determine the molecular weights of polysulfated, -sulfonated, and -phosphorylated biomolecules such as cysteic acid-containing peptides, oligonucleotides, heparin-derived oligosaccharides, and suramin (a drug containing two trisulfonated naphthalene moieties). Peptides and small proteins rich in arginine were used as the basic components. The extent of complex formation correlates with the number of phosphate and sulfate groups in the acidic component and with the number of arginines in the basic component. Neither the acidic amino acid residue aspartic and glutamic acid nor the basic lysine and histidine contribute to complex formation. For oligonucleotides, histone H4 was found to be the best complexing agent investigated. The analytical utility of the complex formation is demonstrated by the molecular-mass determination of acidic compounds from 500 to 6000 Da at the picomole or sub-picomole level with an accuracy of +/- 0.1% or better and by the absence of alkali cation adducts.     

Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones

The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria. We have investigated different effects (i.e. growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (rho degrees ) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH). The data confirmed the importance of oxygen production for doxorubicin toxicity. The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin. Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation. Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants. Epirubicin induced levels of cytotoxicity similar to those of doxorubicin. The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450. GSH did not induce an evident alteration. Neither were genotoxic effects induced. Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants. All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450. Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH. However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation. The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH.     

Identification, characterization and biological activity of oxytocin receptor in the developing human penis

Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT-PCR in human fetal tissues (11th-12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log(10) unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.     

Physicochemical properties and cytopathogenicity of an adenovirus-like agent isolated from corn snake (Elaphe guttata)

Summary A virus isolated from the internal organs of a moribund corn snake (Elaphe guttata) replicated in reptilian cell cultures (IgH-2, TH-1 cells) between 10 and 30°C. Highest infectivity titers of 10^5.5 TCID₅₀/ml were obtained in IgH-2 cells at 25°C. Infected IgH-2 cells showed the development of three morphologically different intranuclear inclusion bodies. During viral assembly the particles formed typical crystalline aggregates in the nucleus. About 64 h after infection progressive desintegration of the nuclear membrane was evident and virus particles were released into the cytoplasm. Different fish cell lines (CLC, CHSE-214, BF-2, PG, RTG-2) were not capable of propagating the virus. The DNA containing agent proved to be stable at pH 3, more or less at pH 12 and to treatment with chloroform, but it was rapidly inactivated at 56°C. Electron microscopy revealed nonenveloped icosahedral particles with a diameter of 65–70 nm.     

Incidental cholecystectomy during colorectal surgery.

OBJECTIVE: To assess the risks and benefits of incidental cholecystectomy in patients having colorectal surgery. SUMMARY BACKGROUND DATA: Cholelithiasis is found commonly during abdominal surgery. Previous studies used disparate methods to assess the risks and benefits of incidental cholecystectomy and have reached contradictory conclusions. METHODS: All patients in whom asymptomatic cholelithiasis was noted during colorectal surgery between January 1982 and December 1986 were studied. Operative morbidity and long-term outcome were assessed by chart review and questionnaire. RESULTS: Three hundred five patients were identified, of whom 195 (63.9%) had an incidental cholecystectomy and 110 (36.1%) did not. The two groups were similar in terms of age, sex, primary disease, and associated medical conditions, although fewer emergency procedures, abdominoperineal resections, and Hartmann's procedures were needed in the cholecystectomy group. The overall operative morbidity rate was the same in both groups. The long-term risk for developing small bowel obstruction was also similar. After a median follow-up of 6 years after hospital discharge, biliary pain or cholecystitis developed in 16 patients (14.6%) in the "no cholecystectomy" group, 12 of whom have had cholecystectomy. Two additional patients had cholecystectomy for acute postoperative cholecystitis while still in the hospital. Six more patients have had incidental cholecystectomy at subsequent laparotomies. The cumulative probability of needing cholecystectomy at 2 and 5 years after the initial colorectal operation was 12.1% and 21.6%, respectively. CONCLUSIONS: Incidental cholecystectomy was not associated with increased postoperative morbidity, whereas the long-term risk that previously asymptomatic gallstones would become symptomatic was substantial. Unless there are clear contraindications, patients with asymptomatic gallstones who have colorectal surgery should have concomitant cholecystectomy.     

An in vivo model of femtosecond laser intrastromal refractive surgery

BACKGROUND AND OBJECTIVE: To develop an animal model for evaluation of femtosecond laser intrastromal refractive surgery. METHODS: Intrastromal photodisruption was performed in New Zealand Albino rabbits using a femtosecond laser system. This surgical pattern consisted of a 100 microm-tick pyramid of laser pulses starting 180 microm below the corneal surface. Animals underwent serial slit lamp examinations and corneal thickness measurements at 1,3,7,14, and 28 days, then monthly up to 1 year. RESULTS: Approximately 70 microm of central corneal thinning were seen at 1 week, remaining stable up to 7 months. CONCLUSIONS: Intrastromal photodisruption with femtosecond lasers produced consistent changes in corneal thickness without loss of corneal transparency. These changes were more stable than those produced with excimer laser procedures in a similar animal model.