Enhanced skin penetration of P20 phosphopeptide using protein transduction domains

Protein transduction domains (PTDs) were recently demonstrated to increase the penetration of the model peptide P20 when the PTD and P20 were covalently attached. Here, we evaluated whether non-covalently linked PTDs were capable of increasing the skin penetration of P20. Two different PTDs were studied: YARA and WLR. Porcine ear skin mounted in a Franz diffusion cell was used to assess the penetration of P20 in the stratum corneum (SC) and viable skin (VS); VS consists of dermis and epidermis without SC. The transdermal delivery of P20 was also assessed. At 1 mM, YARA promoted a 2.33-fold increase in the retention of P20 in the SC but did not significantly increase the amount of P20 that reached VS. WLR significantly increased (2.88-fold) the penetration of P20 in VS. Compared to the non-attached form, the covalently linked WLR fragment was two times more effective in promoting the penetration of P20 into VS. None of the PTDs promoted transdermal delivery of P20 at 4 h post-application. It was concluded that selected non-covalently linked PTDs can be used as a penetration enhancer, but greater skin penetration efficiency can be achieved by covalently binding the PTD to the therapeutic agent. (c) 2007 Elsevier B.V. All rights reserved.

Iontophoretic transport of zinc phthalocyanine tetrasulfonic acid as a tool to improve drug topical delivery

Phthalocyanines have been used as systemic photosensitizers because of their high affinity towards tumour tissue, and the high rates of reactive oxygen species produced when they are irradiated during photodynamic therapy. However, the topical administration of these compounds is limited by their large size, poor hydrosolubility and ionic character. This study aimed to investigate the iontophoretic delivery of charged zinc phthalocyanine tetrasulfonic acid (ZnPcS(4)) from a hydrophilic gel to different skin layers by means of in-vitro and in-vivo studies. Six hours of passive administration was insufficient for ZnPcS(4) to cross the stratum corneum (SC) and to reach the epidermis and dermis. No positive effect was reached when anodal iontophoresis was performed, showing that the drug-electrode attraction effect was higher than the electro-osmosis contribution at a pH of 5.5. Cathodal iontophoresis, however, was able to transport significant amounts of the drug to the viable epidermis. In addition, the absence of NaCl in the formulation significantly increased (by five-fold) the amount of ZnPcS(4) that crossed the SC and accumulated in the epidermis and dermis. It was possible to visualize the drug accumulation in the follicle openings and in the epidermis, even after SC removal. In-vivo experiments in rat skin showed that these results were maintained in an in-vivo model, even with only 15 min of iontophoresis. In addition, confocal analysis of the treated skin showed a homogeneous distribution of ZnPcS(4) in the viable epidermis after this short period of cathodal iontophoresis. Anti-Cancer Drugs 22:783-793 (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Assessment of the percutaneous penetration of cisplatin: The effect of monoolein and the drug skin penetration pathway

It was intended to examine the in vitro penetration of cisplatin (CIS) through porcine skin in the presence of different concentrations of monoolein (MO) as well as to verify the main barrier for CIS skin penetration. In vitro skin penetration of CIS was studied from propylene glycol (PG) solutions containing 0%, 5%, 10%, and 20% of MO using Franz-type diffusion cell and porcine ear skin. Pretreatment experiments with MO and experiments with skin without stratum corneum (SC) were also carried out. Skin penetration studies of CIS showed that the presence of MO doubled the drug permeation through the intact skin. However, permeation studies through the skin without SC caused only a small enhancement of CIS permeation compared to intact skin. Moreover, pretreatment of skin with MO formulations did not show any significant increase in the flux of the drug. In conclusion, MO did not act as a real penetration enhancer for CIS, but it increased the drug partition to the receptor solution improving CIS transdermal permeation. The absence of improvement in drug permeation by MO pretreatment and by the removal of SC indicates that the SC is not the main barrier for the permeation of the metal coordination compound. (c) 2009 Elsevier B.V. All rights reserved.

Evaluation of dermatological effects of cosmetic formulations containing Saccharomyces cerevisiae extract and vitamins

Saccharomyces cerevisiae extract (SCE) is used in cosmetics since it can act in oxidative stress and improve skin conditions. This study investigated dermatological effects of cosmetic formulations containing SCE and/or vitamins A, C and E. The formulation studied was supplemented or not (F1: vehicle) with vitamins A, C and E esters (F2) or with SCE (F3) or with the combination of vitamins and SCE (F4). Formulations were patch tested on back skin of volunteers. For efficacy studies, formulations were applied on volunteers and transepidermal water loss (TEWL), skin moisture (SM), skin microrelief (SMR) and free radicals protection were analysed after 3 h, 15 and 30 days of application. Volunteers were also asked about efficacy perception. It was observed that F4 provoked a slight erythema in one volunteer. All formulations enhanced forearm SM. Only F3 and F4 presented long term effects on SMR and showed higher texture values; F3 had the highest brightness values. Our results suggest that vitamins and SCE showed effects in SM and SMR. Only formulations containing SC had long term effects in the improvement of SMR. Thus, these kinds of evaluations are very important in cosmetics development to evaluate the best risk and benefit correlation. (C) 2008 Elsevier Ltd. All rights reserved.

Distribution of major, minor, and trace metals in lake environments of Antarctica

The concentrations of major, minor and trace metals were measured in water samples collected from five shallow Antarctic lakes (Carezza, Edmonson Point (No 14 and 15a), Inexpressible Island and Tarn Flat) found in Terra Nova Bay (northern Victoria Land, Antarctica) during the Italian Expeditions of 1993-2001. The total concentrations of a large suite of elements (Al, As, Ba, Ca, Cd, Ce, Co, Cr, Cs, Cu, Fe, Ga, Gd, K, La, Li, Mg, Mn, Mo, Na, Nd, Ni, Pb, Pr, Rb, Sc, Si, Sr, Ta, Ti, U, V, Y, W, Zn and Zr) were determined using spectroscopic techniques (ICP-AES, GF-AAS and ICP-MS). The results are similar to those obtained for the freshwater lakes of the Larsemann Hills, East Antarctica, and for the McMurdo Dry Valleys. Principal Component Analysis (PCA) and Cluster Analysis (CA) were performed to identify groups of samples with similar characteristics and to find correlations between the variables. The variability observed within the water samples is closely connected to the sea spray input; hence, it is primarily a consequence of geographical and meteorological factors, such as distance from the ocean and time of year. The trace element levels, in particular those of heavy metals, are very low, suggesting an origin from natural sources rather than from anthropogenic contamination.

Babesia bovis: Genome size, number of chromosomes and telomeric probe hybridisation

Pulsed field gel electrophoresis of intact chromosomes of Babesia bovis revealed four chromosomes in the haploid genome. A telomere probe, derived from Plasmodium berghei, hybridised to eight SfiI restriction fragments of genomic B. bovis DNA digests indicating the presence of four chromosomes. A small subunit (18S) ribosomal RNA gene probe hybridised to the third chromosome only. The genome size of B. bovis is estimated to be 9.4 million base pairs. The sizes of chromosomes 1, 2, 3 and 4 are estimated to be 1.4, 2.0, 2.8 and 3.2 million base pairs, respectively. (C) 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.

Expressed sequenced tags and new genes from the cattle tick, Boophilus microplus

We used the expressed sequenced tags (ESTs) approach to study the genome of the cattle tick Boophilus microplus. One hundred and forty-two of our 234 unique ESTs were from genes not previously identified from ticks, mites or any other arachnids. The largest class of identified ESTs (29%) was from genes involved in transcription and translation. Ninety-one ESTs (39% of all ESTs) did not match any sequences in international databases; some of these may be specific to ticks. Thirteen percent of our ESTs were from ribosomal proteins and two ESTs were for genes implicated in resistance to pesticides. (C) 1998 Chapman & Hall Ltd.

Molecules and morphology in phylogenetic studies of the Hemiuroidea (Digenea : Trematoda : Platyhelminthes)

Phylogenies of trematodes based on characters derived from morphology and life cycles have been controversial. Here, we add molecular data to the phylogenetic study of a group of trematodes, members of the superfamily Hemiuroidea Looss, 1899. DNA sequences from the V4 domain of the nuclear small subunit (18S) rRNA gene and a matrix of morphological characters modified from a previous study were used. There was no significant incongruence between the molecular and the morphological data. However, this was probably due largely to the limited resolving power of the morphological data. Analyses support a monophyletic Hemiuroidea containing at least the families Accacoeliidae, Derogenidae, Didymozoidae, Hirudinellidae, Sclerodistomidae, Syncoeliidae, Isoparorchiidae, Lecithasteridae, and Hemiuridae. These families fall into two principal clades. One contains the first six families and the other the Hemiuridae and lecithasterine lecithasterids. The positions of the hysterolecithine lecithasterids and the Isoparorchiidae were poorly resolved. The Ptychogonimidae may be the sister group of the remaining Hemiuroidea, but there was no support from the molecular data for the placement of the Azygiidae within the superfamily. (C) 1998 Academic Press.

Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance

Acetylcholinesterase is the target of organophosphate and carbamate pesticides. Organophosphate resistance is widespread in the cattle tick, Boophilus microplus, in Australia. We have isolated a cDNA of acetylcholinesterase from B. microplus and show that it would encode a protein 62 kDa in size. The predicted amino acid sequence contains all the residues characteristic of an acetylcholinesterase. Alternative splicing of the transcript was detected at both the 5' and 3' ends. Alternative splicing at the 5' end would result in two proteins differing by six amino acids. This is the first report of alternative splicing of the N-terminal coding region in a cholinesterase. No point mutations were detected in the acetylcholinesterase gene from organophosphate resistant strains of B. microplus. Alternative explanations for resistance to organophosphates in B. microplus are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.

Distinguishing species and populations of Rhipicephaline ticks with its 2 ribosomal RNA

Most populations and some species of ticks of the genera Boophilus (5 spp.) and Rhipicephalus (ca. 75 spp.) cannot be distinguished phenotypically. Moreover, there is doubt about the validity of species in these genera. I studied the entire second internal transcribed spacer (ITS 2) rRNA of 16 populations of rhipicephaline ticks to address these problems: Boophilus,microplus from Australia, Kenya, South Africa and Brazil (4 populations); Boophilus decoloratus from Kenya; Rhipicephalus appendiculatus from Kenya, Zimbabwe and Zambia (7 populations); Rhipicephalus zambesiensis from Zimbabwe (3 populations); and Rhipicephalus evertsi from Kenya. Each of the 16 populations had a unique ITS 2, but most of the nucleotide variation occurred among species and genera. ITS 2 rRNA can be used to distinguish the populations and species of Boophilus and Rhipicephalus studied here. Little support was found for the hypothesis that B. microplus from Australia and South Africa are different species. ITS 2 appears useful for phylogenetic inference in the Rhipicephalinae because in genetic distance, maximum likelihood, and maximum parsimony analyses, most branches leading to species had >95% bootstrap support. Rhipicephalus appendiculatus and R, zambeziensis are closely related, yet their ITS 2 sequences could be distinguished unambiguously. This lends weight to a previous proposal that Rhipicephalus sanguineus and Rhipicephalus turanicus, and Rhipicephalus pumlilio and Rhipicephalus camicasi, respectively, are conspecific, because each of these pairs of species had identical sequences for ca. 250 bp of ITS 2 rRNA.

Inference of phylogeny and taxonomy within the Didymozoidae (Digenea) from the second internal transcribed spacer (ITS2) of ribosomal DNA

DNA sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 11 species from four genera of Didymozoinae (Indodidymozoon, Helicodidymozoon, Rhopalotrema and Neometadidymozoon) and a species of the Lecithasteridae, Lecithaster stellatus. Sequences were used to test the validity of species recognised on morphological criteria and to infer phylogenetic relationships. Sequences of the 11 didymozoids differed by 0.5% to 19%. Our phylogenetic analyses: (i) indicate that species in the genera Helicodidymozoon and Rhopalotrema are a monophyletic group; (ii) support separation of the genus Helicodidymozoon from the genera Indodidymozoon and Neometadidymozoon; and (iii) support recognition of Rhopalotrema as a genus distinct from Neometadidymozoon. We found the gonochoristic species, I. pearsoni and I. suttiei, to be genetically similar to the hermaphroditic species in the genus Indodidymozoon and found no evidence to indicate that they belong in a separate genus.

Synchronization of estrus and fertility in zebu beef heifers treated with three estrus synchronization protocols

The effects on estrus and fertility of 3 estrus synchronization protocols were studied in Brahman beef heifers. In Treatment 1 (PGF protocol; n=234), heifers received 7.5 mg, im prostianol on Day 0 and were inseminated after observed estrus until Day 5. Treatment 2 (10-d NOR protocol; n=220) consisted of norgestomet (NOR; 3 mg, sc implant and 3 mg, im) and estradiol valerate (5 mg, im) treatment on Day -10, NOR implant removal and 400 IU, im PMSG on Day 0, and AI after observed estrus through to Day 5. Treatment 3 (14-d NOR+PGF protocol; n=168) constituted a NOR implant (3 mg, sc) on Day -14, NOR implant removal on Day 0, PGF on Day 16, and AI after observed estrus through to Day 21. All heifers were examined for return to estrus at the next cycle and inseminated after observed estrus. The heifers were then exposed to bulls for at least 21 d. During the period of estrus observation (5 d) after treatment, those heifers treated with the PGF protocol had a lower (P

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the beta-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.

V4 region of small subunit rDNA indicates polyphyly of the Fellodistomidae (Digenea) which is supported by morphology and life-cycle data

There is no morphological synapomorphy for the disparate digeneans, the Fellodistomidae Nicoll, 1909. Although all known life-cycles of the group include bivalves as first intermediate hosts, there is no convincing morphological synapomorphy that can be used to unite the group. Sequences from the V4 region of small subunit (18S) rRNA genes were used to infer phylogenetic relationships among 13 species of Fellodistomidae from four subfamilies and eight species from seven other digenean families: Bivesiculidae; Brachylaimidae; Bucephalidae; Gorgoderidae; Gymnophallidae; Opecoelidae; and Zoogonidae. Outgroup comparison was made initially with an aspidogastrean. Various species from the other digenean families were used as outgroups in subsequent analyses. Three methods of analysis indicated polyphyly of the Fellodistomidae and at least two independent radiations of the subfamilies, such that they were more closely associated with other digeneans than to each other. The Tandanicolinae was monophyletic (100% bootstrap support) and was weakly associated with the Gymnophallidae (< 50-55% bootstrap support). Monophyly of the Baccigerinae was supported with 78-87% bootstrap support, and monophyly of the Zoogonidae + Baccigerinae received 77-86% support. The remaining fellodistomid species, Fellodistomum fellis, F. agnotum and Coomera brayi (Fellodistominae) plus Proctoeces maculatus and Complexobursa sp. (Proctoecinae), formed a separate clade with 74-92% bootstrap support. On the basis of molecular, morphological and life-cycle evidence, the subfamilies Baccigerinae and Tandanicolinae are removed from the Fellodistomidae and promoted to familial status. The Baccigerinae is promoted under the senior synonym Faustulidae Poche, 1926, and the Echinobrevicecinae Dronen, Blend & McEachran, 1994 is synonymised with the Faustulidae. Consequently, species that were formerly in the Fellodistomidae are now distributed in three families: Fellodistomidae; Faustulidae (syn. Baccigerinae Yamaguti, 1954); and Tandanicolidae Johnston, 1927. We infer that the use of bivalves as intermediate hosts by this broad range of families indicates multiple host-switching events within the radiation of the Digenea.