Distribution of specific keratin-associated beta-proteins (beta-keratins) in the epidermis of the lizard Anolis carolinensis helps to clarify the process of cornification in lepidosaurians.

The epidermis of different scales in the lizard Anolis carolinensis expresses specific keratin-associated beta-proteins (beta-keratins). In order to localize the sites of accumulation of different beta-proteins, we have utilized antibodies directed against representative members of the main families of beta-proteins, the glycine-rich (HgG5), glycine-cysteine rich (HgGC3), glycine-cysteine medium-rich (HgGC10), and cysteine-rich (HgC1) beta-proteins. Immunoblotting and immunocytochemical controls confirm the specificity of the antibodies made against these proteins. Light and ultrastructural immunocytochemistry shows that the glycine-rich protein HgG5 is present in beta-layers of different body scales but is scarce in the oberhautchen and claws, and is absent in alpha-layers and adhesive setae. The cysteine-glycine-rich protein HgGC3 is low to absent in the oberhautchen, beta-layer, and mesos-layer but increases in alpha-layers. This beta-protein is low in claws where it is likely associated with the hard alpha-keratins previously studied in this lizard. The glycine-cysteine medium-rich HgGC10 protein is low in the beta-layer, higher in alpha-layers, and in the oberhautchen. This protein forms a major component of setal proteins including those of the adhesive spatula that allow this lizard to stick on vertical surfaces. HgC1 is poorly localized in most epidermis analyzed including adhesive setae and claws and appears as a minor component of the alpha-layers. In conclusion, the present study suggests that beta- and alpha-layers of lizard epidermis represent regions with different accumulation of glycine-rich proteins (mainly for mechanical resistance and hydrophobicity in the beta-layer) or cysteine-glycine-rich proteins (for both resistance and elasticity in both alpha- and beta-layers).

Is carotid endarterectomy in octogenarians more dangerous than in younger patients?

AIM: The risk for developing stroke increases with the advancing age, peaking over age 80. In elderly patients, carotid endarterectomy may provide prophylaxis against stroke. Aim of our study was to compare patients 80 years or older with patients younger than 80 undergoing carotid endarterectomy. Endpoints were perioperative mortality and morbidity. METHODS: From January 1996 to December 2002, 1 659 patients underwent a 1 733 carotid endarterectomy for a symptomatic or asymptomatic significant carotid lesion. Among them, 125 patients were 80 years or older. We analyzed death and stroke rate from cerebrovascular accidents, TIA as well as non cerebrovascular complications and death rate postoperatively and in the long term follow-up. The Pearson's chi-squared(2) test was used for the statistical analysis on risk factors, morbidity and mortality. The Log rank test was used for cumulative stroke-free and survival rates between the 2 groups (level of confidence p<0.05). RESULTS: Risk factors were similar in both groups. No statistical difference was observed in the stroke, TIA, mortality and stroke free rates between the 2 groups. CONCLUSIONS: The results of our study show that perioperative and postoperative mortality and morbidity as well as the long-term stroke-free rate does not differ significantly in patients 80 years or older compared to patients younger than 80 undergoing carotid endarterectomy.

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.

Polylysine-porphycene conjugates as efficient photosensitizers for the inactivation of microbial pathogens.

Porphycenes are electronic isomers of porphyrins which, when neutral, display no appreciable photosensitizing action towards Gram-negative bacteria. The covalent binding of oligomeric polylysine moieties, which are cationic at physiological pH values, endows porphycenes with a significant phototoxic activity against Gram-negative bacteria while retaining their photoefficiency against a variety of microbial pathogens, including Gram-positive bacteria, fungi and mycoplasmas. The effect of the polylysine moiety is dependent on both the polylysine concentration and the degree of oligomerization. A suitable interplay among the various parameters opens the possibility to obtain either a broad spectrum of antimicrobial activity or a selective action toward a specific pathogen while minimizing the damage to human fibroblasts.

Effect of chemical structure and hydrophobicity on the pharmacokinetic properties of porphycenes in tumour-bearing mice.

The efficiency and selectivity of tumour targeting by several tetra-n-propylporphycene (TPPn) and tetrakis(methoxyethyl)porphycene (TMPn) derivatives have been studied by administering 3.76 micromol/kg of aqueous or liposomal porphycene formulations to BALB/c mice bearing an i.m. implanted MS-2 fibrosarcoma. These 2 parameters have been studied as a function of the type of substituents linked to the 9-position of the macrocycle by amide, ester or ether functional groups. The pharmacokinetic properties appear to be controlled mainly by the degree of porphycene hydrophobicity, as evaluated by measuring their retention times in a C 18 column for HPLC. Thus, the post-injection time (T50) at which the porphycene concentration in the plasma decreases to 50% of the initial value ranged from a few minutes for the less hydrophobic to several hours for the more hydrophobic porphycenes. An increase in hydrophobicity also was accompanied by an enhanced efficiency and selectivity of tumour targeting. The less hydrophobic porphycenes showed a maximum tumour uptake of 0.5-2 nmol/g of tissue at 10-20 min after administration with a tumour/peri-tumoural concentration ratio around 2-3, while those with higher hydrophobicity reached tumour concentrations of 7-8 nmol/g at 24-48 hr after administration with concentration ratios higher than 20.

Laser-induced fluorescence studies of the biodistribution of carotenoporphyrins in mice.

The biodistribution of two recently developed tumour markers, trimethylated (CP(Me)3) and trimethoxylated (CP(OMe)3) carotenoporphyrin, was investigated by means of laser-induced fluorescence (LIF) after i.v. injection into 38 tumour-bearing (MS-2 fibrosarcoma) female Balb/c mice. At 3, 24, 48 or 96 h after administration, the carotenoporphyrin fluorescence was measured in tumoral and peritumoral tissue, as well as in the abdominal, thoracic and cranial cavities. The fluorescence was induced by a nitrogen laser-pumped dye laser, emitting light at 425 nm, and analysed by a polychromator equipped with an image-intensified CCD camera. The fluorescence was evaluated at 490, 655 and 720 nm: the second and third wavelengths represent the carotenoporphyrin (CP)-related peaks, whereas the first one is close to the peak of the tissue autofluorescence. The tumour and the liver were the two tissue types showing the strongest carotenoporphyrin-related fluorescence, whereas the cerebral cortex and muscle consistently exhibited weak substance-related fluorescence. In most tissue types, the fluorescence intensities decreased over time. A few exceptions were observed, notably the liver, in which the intensity remained remarkably constant over the time period investigated.

Liposome-delivered 131I-labelled Zn(II)-phthalocyanine as a radiodiagnostic agent for tumours.

131I-Zn(II)-phthalocyanine (ZnPc) incorporated into unilamellar liposomes has been systemically injected to mice bearing a transplanted MS-2 fibrosarcoma. Biodistribution studies show that the pharmacokinetic behaviour of 131I-ZnPc is very similar to that defined for the parent molecule ZnPc including a serum half-life of ca. 12 h, a high recovery from liver and spleen and minimal accumulation in kidney and brain. The most important pharmacokinetic parameter is represented by the high tumour/ muscle ratio of 131I-ZnPc concentration (ca. 9 at 24 h post-injection). These results suggest the possible use of the radiolabelled derivative for a real-time non-invasive monitoring of the ZnPc concentration in the tumour and peritumoural tissue during photodynamic therapy.

Laser-induced fluorescence in malignant and normal tissue in mice injected with two different carotenoporphyrins.

Laser-induced fluorescence (LIF) was used to characterise the localisation of an intravenously administered trimethylated carotenoporphyrin [CP(Me)3] and a trimethoxylated carotenoporphyrin [CP(OMe)3] in an intramuscularly transplanted malignant tumour (MS-2 fibrosarcoma) and healthy muscle in female Balb/c mice, 3, 24, 48 and 96 h post injection. The fluorescence was induced with a dye laser pumped by a nitrogen laser, emitting light at 425 nm. The fluorescence spectra were recorded in the region 455-760 nm using a polychromator equipped with an image-intensified CCD camera. The tumour/peritumoral muscle ratio was about 5:1 for CP(Me)3 and about 6:1 for CP(OMe)3 in terms of the background-free fluorescence intensity, which peaked at about 655 nm. By including the endogenous tissue fluorescence, the contrast was further enhanced by a factor of approximately 2.

CGP 55398, a liposomal Ge(IV) phthalocyanine bearing two axially ligated cholesterol moieties: a new potential agent for photodynamic therapy of tumours.

Ge(IV) phthalocyanine (GePc) with two axially ligated cholesterol moieties was prepared by chemical synthesis and incorporated in a monomeric state into small unilamellar liposomes (CGP 55398). Upon photoexcitation with light wavelengths around its intense absorption peak at 680 nm, GePc shows an efficient photosensitising activity towards biological substrates through a mechanism which largely involves the intermediacy of singlet oxygen. GePc injected systemically into mice bearing an intramuscularly implanted MS-2 fibrosarcoma is quantitatively transferred to serum lipoproteins and localises in the tumour tissue with good efficiency: at 24 h post injection the GePc content in the tumour is 0.74 and 1.87 micrograms per g of tissue with a tumour/peritumoral ratio of 4.35 and 5.67 for injected doses of 0.76 and 1.52 mg kg-1 respectively. At this time the red-light irradiation of the GePc-loaded fibrosarcoma causes a fast and massive tumour necrosis involving both malignant cells and blood vessels.

Carotenoporphyrins as selective photodiagnostic agents for tumours.

The covalent binding of a carotene moiety to one phenyl ring and meso-tetraphenyl-substituted porphyrins (see Figure 1) efficiently quenches the photosensitising activity of the porphyrin while a relatively large yield of fluorescence emission around 650 nm is retained. Pharmacokinetic studies performed with two carotenoporphyrins (CPs) and the corresponding porphyrins (Ps) in Balb/c mice bearing an MS-2 fibrosarcoma show that the two Ps give a high selectivity of tumour localisation (tumour/peritumoral tissue ratios of dye concentration ranging between c. 30 and 90 at 24 h after injection of 4.2-8.4 mumol kg-1 in a Cremophor emulsion) and photosensitive tumour necrosis upon red light irradiation. For the same injected doses, the two CPs show no tumour-photosensitising activity even though they localise in the tumour in concentrations of the order of 10-40 micrograms g-1 at 24 h with tumour/peritumoral ratios larger than 10. Thus, the fluorescence emitted by these CPs in the tumour can be used for photodiagnostic purposes with no risk of skin photosensitisation. However, this approach is presently limited by the large accumulation and prolonged retention of the CPs in the liver and spleen.