Photodynamic laser cyclodestruction with chloroaluminum sulfonated Phthalocyanine (CASPc) or photofrin®(PII) Vs. Nd:YAG laser cyclodestruction in a pigmented rabbit model

Background and Objective: To investigate Photofrin® (PII) and CASPc for photodynamic therapy (PDT) of the ciliary body in rabbits. Study Design/Materials and Methods: PII (10 mg/kg) or CASPc (1 mg/kg) was given by ear vein. Pharmacokinetics were studied in frozen sections by fluorescence microscopy (CCD camera based low light detection system with digital image processing) at 1 and 24 h (8 rabbits;16 eyes). Laser light was delivered (argon pumped dye laser;630 and 675 nm;8 rabbits;16 eyes) by contact fiberoptic. To compensate for iris attenuation, irradiance was 125 mW/cm² (20, 40, 80, or 160 J/cm²). Controls (4 rabbits;8 eyes) received laser light without photochemicals (OD) and for comparison, continuous wave Nd:YAG laser by fiberoptic (0.8–1.2J;OS). Results: Localization studies showed intravascular distribution with some selective ciliary body distribution at 24 h (PII > CASPc). Rabbits treated with PII or CASPc exhibited variable amounts of gross ciliary body edema, infarction, and necrosis by 24–48 h. This response was not seen in PDT control tissues;damage was seen in the iris and ciliary body, with partial vacuolization of the pigment epithelium. Conclusion: PDT may offer a more selective approach to ciliary body destruction. A small but significant thermal effect was seen during PDT from melanin photon uptake with damage to iris and ciliary body. Thermal damage and potential interaction with ocular visual pigments may limit use of these photochemicals and wavelengths for PDT of the ciliary body © 1995 Wiley-Liss, Inc.     

The retinae of Prototherian mammals possess neuronal types that are characteristic of non-mammalian retinae

This study has shown that the retinae of Prototherian (egg-laying) mammals possess two neuronal types that are present in non-mammalian retinae, but absent or morphologically different in the retinae of Eutherian (placental) mammals. First, endogenous serotonin-like immunoreactivity has been localized in a population of presumptive amacrine cells in the platypus retina, the first such report in a mammalian retina. Second, the protein kinase C-immunoreactive (PKC-IR) bipolar cells in the echidna retina appear similar to the PKC-IR bipolars in the chicken retina, in that their dendrites give rise to a Landolt's club and their axons are multistratified. By contrast, the PKC-IR rod bipolar cells in the rabbit and in the brushtail possum, a Metatherian (marsupial) mammal, have no Landolt's clubs and their axons form terminal lobes in the innermost stratum of the inner plexiform layer.     

Detection of Bacteroides fragilis Enterotoxin Gene by PCR

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.     

Transforming Growth Factor-β1 Induces Apoptosis through Down-Regulation of c-mycGene and Overexpression of p27Protein in Cervical Carcinoma

Transforming growth factor-β1 (TGF-β1) is known to be a potent growth inhibitor for many cell types, including most epithelial cells. In skin keratinocytes, TGF-β1 has been shown to inhibit growth and to rapidly reduce c-mycexpression. However, the molecular mechanism of TGF-β1 action on cell growth of cervical carcinoma has not yet been elucidated. We thus assessed the effect of TGF-β1 on the growth of cervical carcinoma cell lines. Two cervical squamous carcinoma cell lines, CUMC-3 and CUMC-6, were incubated with varying concentrations of TGF-β1, and growth inhibition was evaluated with tetrazolium-based colorimetric assay. After culture in TGF-β1 for 24 h, inhibition of growth was detected in a dose-dependent manner at concentrations of 0.1–10 ng/ml in both cell lines. This effect of TGF-β1 on cultured carcinoma cells was associated with apoptotic process including oligonucleosomal ladder DNA and apoptotic body formations. Northern blot analysis revealed c-mycmRNA expression was suppressed by 10 ng/ml of TGF-β1 following 3 h of treatment in both cell lines. Western blot analysis showed that the level of p27^Kip1protein was increased after TGF-β1 treatment in both cell lines. These results suggest that the mechanisms by which TGF-β1 inhibits the growth of cervical carcinoma are complex and may include effects on down-regulation of c-mycgene, and overexpression of p27^Kip1protein.     

Total synthesis of the four stereoisomers of dihexadecanoyl phosphatidylinositol and the substrate stereospecificity of human erythrocyte membrane phosphatidylinositol 4-kinase

A new and convenient method for the preparation of the four stereoisomers of dihexadecanoyl phosphatidylinositol has been developed. An enantiomeric pair of acid-labile, pentaprotected myo-inositol building blocks was synthesized in high yield and coupled with chiral phenyl dihexadecanoylglyceryl phosphates to give the fully protected phosphatidylinositols. These were subsequently deprotected by hydrogenolysis and self-hydrolysis in aqueous ethanol to give the desired pure products. Comparison of these compounds as potential substrates for a partially purified phosphatidylinositol 4-kinase (EC 2.7.1.67) derived from human erythrocyte membranes revealed that the chirality of the inositol ring is crucial for efficient phosphorylation, whereas the chirality of the glycerol moiety is relatively unimportant. Moreover, the similarity in phosphorylation rates of the naturally occurring mammalian phospholipid, I, and its synthetic stereochemical counterpart, compound 10a, suggests that the enzyme is relatively tolerant to changes in fatty acid composition.