Differential mechanisms for calcium-dependent protein/membrane association as evidenced from SPR-binding studies on supported biomimetic membranes

In this work, two different types of supported biomimetic membranes were designed to study the membrane binding properties of two different proteins that both interact with cellular membranes in a calcium-dependent manner. The first one, neurocalcin, is a member of a subfamilly of EF-hand calcium-binding proteins that exhibit a calcium-myristoyl switch. The second protein is a bacterial toxin, the adenylate cyclase produced by Bordetella pertussis, the causative agent of whooping cough. The biomimetic membranes constructed in this study were either hybrid bilayer membranes or polymer-tethered membranes. Hemimembrane formation was obtained in two steps: a monolayer of 1-octadecanethiol or octadecyltrichlorosilane was self-assembled on top of the gold or glass surface, respectively, and then the egg-phosphatidyl choline (PC) vesicle fused on the hydrophobic alkyl layer. Polymer-tethered membranes on solid support were obtained using N-hydroxysuccinimide (NHS)-terminated-poly(ethyleneglycol) (PEG)-phospholipids as anchoring molecules. Egg-PC/1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-poly(ethyleneglycol)-N-hydroxy-succinimide (DSPE-PEG-NHS) mixture liposomes were injected on the top of an amine grafted surface (cysteamine-coated gold or silanized glass); vesicles were linked to the surface and disrupted, leading to the formation of a bilayer. The biomimetic membrane constructions were followed by surface plasmon spectroscopy, while membrane fluidity and continuity were observed by fluorescence microscopy. Protein/membrane binding properties were determined by resonance surface plasmon measurements. The tethered bilayer, designed here, is very versatile as it can be adapted easily to different types of support. The results demonstrate the potentialities of such polymer-tethered artificial membranes for the study of proteins that insert into biological membranes such as toxins and/or integral membrane proteins.     

Differential modulation of dopaminergic systems in the rat brain by dietary protein

Rats that consume a diet 50% rich in protein exhibit hyperactivity and hyperresponsiveness to nociceptive stimuli, in which facilitation of dopaminergic activity has been implicated. We studied the regional changes in the concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brains of rats that were maintained on high-protein (HP, 50% casein), normal-protein (NP, 20% casein), and low-protein (LP, 8% casein) diets for 36 weeks. Brain nuclei that represented different DAergic systems were punchdissected and analyzed using HPLC. In the substantia nigra, the striatum, and the dentate gyrus, DA concentrations decreased and increased, respectively, with a decrease and increase in dietary protein (p<0.05 compared to the NP diet). Similar trends in the effect of the HP diet were observed in the ventral tegmental area, amygdala, frontal cortex, subiculum, centromedial nucleus (CM) of the thalamus, and inferior colliculi (IC), although the differences in DA concentrations were not statistically significant. These brain areas also showed a pattern of decreased DA concentration in association with the LP diet, and the differences were statistically significant (p<0.05) in the CM and IC. DA concentrations in most regions of the midbrain and brainstem were not different between the diet groups, nor were consistent trends observed in those regions. Also, there were no consistent relationships between DOPAC/DA and HVA/DA ratios and dietary protein level. These data suggest that only discrete dopaminergic neuronal circuits in the rat forebrain were sensitive to changes in dietary protein level.     

Origin and phylogenetic distribution of Alu DNA repeats: irreversible events in the evolution of primates.

Over the past 60 million years, or so, approximately one million copies of Alu DNA repeats have accumulated in the genome of primates, in what appears to be an ongoing process. We determined the phylogenetic distribution of specific Alu (and other) DNA repeats in the genome of several primates: human, chimpanzee, gorilla, orangutan, baboon, rhesus, and macaque. At the population level studied, the majority of the repeats was found to be fixed in the primate species. Our data suggest that new Alu elements arise in unique, irreversible events, in a mechanism that seems to preclude precise excision and loss. The same insertions did not arise independently in two species. Once inserted and genetically fixed, the DNA elements are retained in all descendant lineages. The irreversible expansion of Alu s introduces a vector of time into the evolutionary process, and provides realistic (rather than statistical) answers to questions on phylogenies. In contrast to point mutations, the present distribution of individual Alu s is congruent with just one phylogeny. We submit that only irreversible and taxonomically relevant events are at the molecular basis of evolution. Most point mutations do not belong to this category.     

Use of caffeic acid phenethyl ester and cortisone may prevent proliferative vitreoretinopathy

PURPOSE: To investigate whether caffeic acid phenethyl ester (CAPE) and cortisone prevent proliferative vitreoretinopathy (PVR). METHODS: Twenty pigmented rabbits were used in this study. All rabbits except controls received an intravitreal injection of 0.15 ml (75,000 U) of platelet-rich plasma into their left eye. The animals were divided into four groups: group I was treated with intraperitoneal injection of 0.5 ml (15 micromol/kg) of CAPE for 3 days, group II received 0.15 ml (4 mg/kg) of intravitreal cortisone, group III received nothing (blank group), and group IV (control group) received only 1 ml of 1% ethanol intraperitoneally daily for 3 days. Proliferative changes were graded in a masked fashion by indirect ophthalmoscopy for a 15-day follow-up period. The malondialdehyde (MDA), reduced glutathione (GSH) and total nitrite (NO) levels were measured in the vitreous humor. RESULTS: The grades of PVR were B-C in group I, and C-D in group II. The PVR grade in the control group was C-D. The mean MDA level in group I (4.0+/-0.8 micromol/l) was significantly lower than in the blank group (6.0 micromol/l) (p < 0.05). The mean GSH level in group I (71.0+/-11.2 micromol/l) was significantly different than in the blank group (p < 0.05). The MDA and GSH levels in group II were 4.7+/-0.6 micromol/l and 53.8+/-7.8 micromol/l, respectively. Both these levels were not significantly different from the blank group (p > 0.05). The NO levels in both treatment groups were significantly lower than in the blank group (p < 0.001). CONCLUSION: These findings suggest an inhibitory effect of CAPE on PVR. The inhibitory effect was supported by lower MDA and NO with higher GSH levels in treatment groups than in the blank group. There was no detected significant effect of cortisone for preventing PVR experimentally.     

Bilateral autogenous breast reconstruction using perforator free flaps: a single center's experience

The authors present a single center's experience in bilateral breast reconstruction using perforator free flaps. The aim of this study was to show their indications, surgical technique, and results. A series of 53 patients underwent this procedure between February of 1996 and October of 2002. The surgical procedures were performed on patients with bilateral breast cancer (11 patients), patients with unilateral breast cancer and contralateral prophylactic mastectomy (22 patients), patients who had undergone bilateral prophylactic mastectomy (18 patients), a patient with Poland's syndrome, and a patient whose aesthetic breast augmentation had failed. Primary and secondary bilateral breast reconstructions were done in 18 and four patients, respectively. Eighteen patients who had earlier undergone breast reconstruction with implants had a tertiary breast reconstruction. Combined reconstruction (primary with secondary and primary with tertiary reconstruction) was done in 13 patients. Ninety-eight deep inferior epigastric perforator flaps and eight superior gluteal artery perforator flaps were used. The average operative time was 10 hours (range, 8 to 14.5 hours) for the simultaneous bilateral reconstruction. Total flap necrosis occurred in two cases (one deep inferior epigastric perforator flap and one superior gluteal artery perforator flap). Partial flap necrosis was not encountered, and fat necrosis was found in one deep inferior epigastric perforator flap (1 percent). Two pulmonary infections, one deep vein thrombosis, and one cardiac arrhythmia occurred as postoperative complications. The mean hospital stay was 9 days (range, 6 to 20 days). Abdominal bulging was reported in one patient. There were no recurrent disease or cancer manifestations, with an average follow-up of 3.5 years. This series clearly shows that perforator flaps are reliable and useful tools for bilateral breast reconstruction. This technique decreases the donor-site morbidity and offers an excellent aesthetic and long-term outcome and high patient satisfaction.     

Clinical features, epidemiology, and efficacy and safety of intralesional antimony treatment of cutaneous leishmaniasis: recent experience in Turkey

A total of 1,030 patients, 40.2% men and 59.8% women, identified during the period of October 1998 to November 2002 as having cutaneous leishmaniasis (CL), were studied; 1,431 lesions were identified in the 1,030 patients. One lesion was present in 80.7% of the patients. The size of the lesions (longest axis) was 13.6 mm (standard, 12.1 mm; range 3-150 mm). Most of the lesions were of the papular type (51.2%), although several atypical clinical presentations of CL were observed. The duration of the disease ranged between 1 and 72 mo (mean duration, 10.8 mo). The clinical suspicion of CL was confirmed by the observation of amastigotes on lesion tissue samples stained by Giemsa. The test was positive in 851 of 1,030 patients (82.6%). Intralesional meglumine antimonate solution (85 mg Sb/ml, 0.2-1 ml, depending on the size of the lesion) weekly until complete cure or up to 20 wk was used for first-line therapy of 890 patients (86.4%). We found that this regimen of intralesional Sb has an efficacy of 97.2% with a low relapse rate of 3.9% and no serious adverse side effects.     

Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications

AIMS: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. METHODS AND RESULTS: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8-10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45.4 and 163.6 U g(-1) protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35-38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4.5-5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6-7, in a large temperature range (30-70 degrees C), and was stable below 60 degrees C. The main kinetic constants were determined. The tyrosinase was able to convert p-tyrosol and p-coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. CONCLUSIONS: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg(-1) protein for monophenolase and diphenolase respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking.