Granular gland transcriptomes in stimulated amphibian skin secretions.

Amphibian defensive skin secretions are complex, species-specific cocktails of biologically active molecules, including many uncharacterized peptides. The study of such secretions for novel peptide discovery is time-limited, as amphibians are in rapid global decline. While secretion proteome analysis is non-lethal, transcriptome analysis has until now required killing of specimens prior to skin dissection for cDNA library construction. Here we present the discovery that polyadenylated mRNAs encoding dermal granular gland peptides are present in defensive skin secretions, stabilized by endogenous nucleic acid-binding amphipathic peptides. Thus parallel secretory proteome and transcriptome analyses can be performed without killing the specimen in this model amphibian system--a finding that has important implications in conservation of biodiversity within this threatened vertebrate taxon and whose mechanistics may have broader implications in biomolecular science.

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8 - a novel prokineticin from Bombina orientalis

Prokineticins are small (8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr = 7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.

Unmasking venom gland transcriptomes in reptile venoms

While structural studies of reptile venom toxins can be achieved using lyophilized venom samples, until now the cloning of precursor cDNAs required sacrifice of the specimen for dissection of the venom glands. Here we describe a simple and rapid technique that unmasks venom protein mRNAs present in lyophilized venom samples. To illustrate the technique we have RT-PCR-amplified a range of venom protein transcripts from cDNA libraries derived from the venoms of a hemotoxic snake, the Chinese copperhead (Deinagkistrodon acutus), a neurotoxic snake, the black mamba (Dendroaspis polylepis), and a venomous lizard, the Gila monster (Heloderma suspectum). These include a metalloproteinase and phospholipase A2 from D. acutus, a potassium channel blocker, dendrotoxin K, from D. polylepis, and exendin-4 from H. suspectum. These findings imply that the apparent absence and/or lability of mRNA in complex biological matrices is not always real and paves the way for accelerated acquisition of molecular genetic data on venom toxins for scientific and potential therapeutic purposes without sacrifice of endangered herpetofauna.

Optimisation of radiotherapy for carcinoma of the parotid gland: a comparison of conventional, three-dimensional conformal, and intensity-modulated techniques

Background and purpose: To compare external beam radiotherapy techniques for parotid gland tumours using conventional radiotherapy (RT), three-dimensional conformal radiotherapy (3DCRT), and intensity-modulated radiotherapy (IMRT). To optimise the IMRT techniques, and to produce an IMRT class solution.Materials and methods: The planning target volume (PTV), contra-lateral parotid gland, oral cavity, brain-stem, brain and cochlea were outlined on CT planning scans of six patients with parotid gland tumours. Optimised conventional RT and 3DCRT plans were created and compared with inverse-planned IMRT dose distributions using dose-volume histograms. The aim was to reduce the radiation dose to organs at risk and improve the PTV dose distribution. A beam-direction optimisation algorithm was used to improve the dose distribution of the IMRT plans, and a class solution for parotid gland IMRT was investigated.Results: 3DCRT plans produced an equivalent PTV irradiation and reduced the dose to the cochlea, oral cavity, brain, and other normal tissues compared with conventional RT. IMRT further reduced the radiation dose to the cochlea and oral cavity compared with 3DCRT. For nine- and seven-field IMRT techniques, there was an increase in low-dose radiation to non-target tissue and the contra-lateral parotid gland. IMRT plans produced using three to five optimised intensity-modulated beam directions maintained the advantages of the more complex IMRT plans, and reduced the contra-lateral parotid gland dose to acceptable levels. Three- and four-field non-coplanar beam arrangements increased the volume of brain irradiated, and increased PTV dose inhomogeneity. A four-field class solution consisting of paired ipsilateral coplanar anterior and posterior oblique beams (15, 45, 145 and 170o from the anterior plane) was developed which maintained the benefits without the complexity of individual patient optimisation.Conclusions: For patients with parotid gland tumours, reduction in the radiation dose to critical normal tissues was demonstrated with 3DCRT compared with conventional RT. IMRT produced a further reduction in the dose to the cochlea and oral cavity. With nine and seven fields, the dose to the contra-lateral parotid gland was increased, but this was avoided by optimisation of the beam directions. The benefits of IMRT were maintained with three or four fields when the beam angles were optimised, but were also achieved using a four-field class solution. Clinical trials are required to confirm the clinical benefits of these improved dose distributions.

T-cell-rich histiocyte-rich B-cell lymphoma of parotid gland

We describe a malignant lymphoma of the parotid gland arising in a patient with Sjögren's syndrome. Diagnosis was established on needle biopsy which showed a mixed population of lymphoid cells. Immunohistochemistry revealed B-lymphoid cells, T-lymphoid cells and histiocytes. Clonal immunoglobulin heavy chain gene rearrangement was demonstrated using the polymerase chain reaction. Within the confines of the small biopsy, the lesion qualifies for the designation T-cell-rich histiocyte-rich B-cell lymphoma. The value of molecular techniques in the diagnosis of malignant lymphoma on limited tissue samples is highlighted by this case.