High dose irradiation after pleurectomy/decortication or biopsy for pleural mesothelioma treatment.

PURPOSE: The role played by radiation therapy after pleurectomy/decortication or surgical biopsy in malignant pleural mesothelioma is uncertain. We treated patients with accelerated hypofractionated radiotherapy using helical tomotherapy and intensity-modulated arc therapy in an attempt to keep lung toxicity to a minimum. The present study reports the feasibility and toxicity of this approach. MATERIAL AND METHODS: Between 2008 and 2012, 36 patients with malignant pleural mesothelioma underwent accelerated hypofractionated radiotherapy to the hemithorax after pleurectomy/decortication (19 patients) or biopsy (17 patients). The prescription dose was 25Gy in five fractions over 5 consecutive days. RESULTS: We observed three patients with G3 pneumonitis, five cases of grade 2 dyspnea and six cases of grade 2 cough. The median follow-up was 37 months (range: 3-54 months). The median overall survival for patients who underwent pleurectomy/decortication followed by radiotherapy was 21.6 months [95% confidence interval (95% CI): 15.5-24.1] compared to 19.4 months for patients not submitted to surgery. CONCLUSION: Treatment of intact lung with pleural intensity-modulated arc irradiation in malignant pleural mesothelioma patients with malignant pleural mesothelioma proved safe and feasible, with an acceptable rate of pneumonitis. Survival rates were encouraging for both biopsy-only and pleurectomy/decortication groups. We are currently conducting a phase II dose escalation trial in a similar patient setting to prospectively evaluate the impact of radiotherapy on toxicity, disease-free survival and overall survival.

Molecular cloning and sequence determination of a novel aspartic proteinase from Antarctic fish.

In the present report, we describe a novel aspartic proteinase from the liver of two Antarctic fish species. The nucleotide sequences of the cDNA obtained from the two fishes show 90% identity with each other but only 58% identity with aspartic proteinases from other sources. Sequence analysis shows features for the Antarctic enzymes which are not present in related enzymes of other organisms.

Cathepsin D from the liver of the antarctic icefish Chionodraco hamatus exhibits unusual activity and stability at high temperatures1.

Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion-exchange chromatography followed by affinity chromatography on concanavalin-A Sepharose. The purified enzyme showed a molecular mass of 40 kDa and displayed optimal activity at pH 3.0 with a synthetic chromogenic substrate. The N-terminal sequence of this proteinase was determined by automated Edman degradation and was used to design a primer for use in reverse-transcriptase polymerase chain reaction. The open reading frame of the cloned cDNA encoded an aspartic proteinase, which contained the experimentally determined N-terminal sequence. The predicted sequence (396 residues) had a high similarity with those of cathepsin D from various vertebrate sources, but was considerably different from that of nothepsin, a distinct aspartic proteinase described previously from Antarctic fish [1]. Determination of kinetic parameters for substrate hydrolysis showed that, at temperatures between 8 and 50 degrees C, the icefish cathepsin D had a higher specificity constant (kcat/Km) than human cathepsin D. The stability of both enzymes was measured at 50 degrees C and half-lives of 55 and 3 min were derived for icefish and human cathepsin D, respectively.

Identification of cadmium-sensitive genes in the Antarctic fish Chionodraco hamatus by messenger RNA differential display.

To investigate the ability of cadmium to affect gene transcription in fish, the messenger RNA (mRNA) differential display technique was used to analyze gene expression in the Antarctic icefish Chionodraco hamatus exposed to sublethal doses of cadmium salt. Seven DNA complementary to RNA (cDNA) bands whose steady-state levels of expression significantly changed in response to cadmium exposure were identified. The results obtained show that two groups of genes are affected by cadmium in icefish liver. The first group comprises genes that are up-regulated by the metal: in particular, a gene encoding the heat-shock protein HSP70 and another encoding a protein homologous to GP49 of Sparus aurata egg envelope. The other group comprises genes down-regulated by cadmium. These are the transferrin gene and a gene encoding a protein presenting homology to mouse T2K, a kinase having a role in the prevention of apoptosis. Three cDNAs had no homology to known gene sequences, thus suggesting that may either encode not yet identified proteins, or correspond to untranslated regions of mRNA molecules.

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish).

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT-PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.

Molecular cloning, sequence and expression of follicle-stimulating hormone receptor in the lizard Podarcis sicula.

The present paper reports the full nucleotide sequence of a cloned cDNA prepared from RNA of lizard ovaries. The open reading frame consists of 2019 nucleotides, which encodes a protein of 673 amino acids belonging to the G protein-coupled receptor superfamily with a large extracellular N-terminal domain involved in hormone recognition. The transmembrane domain ends with a short intracytoplasmic COOH-terminal domain involved in effector activation. Phylogenetic analysis showed that the lizard receptor belongs to the family of follicle-stimulating hormone (FSH) receptors. The hydrophobicity profile is similar to that observed for mammalian and avian FSH receptors. Northern blot analysis of total RNA revealed that the FSH receptor is expressed at high levels in the ovary. In situ hybridization experiments demonstrate that FSH receptor mRNA is specifically localized within the small cells of the follicular epithelium surrounding the oocyte.

Structural and functional analysis of metal regulatory elements in the promoter region of genes encoding metallothionein isoforms in the Antarctic fish Chionodraco hamatus (icefish).

To investigate the regulation of Chionodraco hamatus metallothionein (MT) encoding genes about 1000-bp regions of both MT-I and MT-II gene promoters were cloned and sequenced. Both promoters were rich in A-T content, and lacked the canonical TATA box; several putative cis-regulatory sequences were also present. In the MT-I promoter, four MREs were identified within the first 300 bp from the ATG codon. In the MT-II promoter, seven MREs were organized into two clusters, one containing three MREs located close to the ATG codon, and the other consisting of four MREs lying 500-900 bp upstream of the transcription starting point. The alignment of the MT-I and MT-II promoter regions showed 57% identity, which increased to 87% in the 300-bp region upstream of the ATG. Only the three proximal putative MREs identified were conserved both in position and sequence. Functional analysis of MT-I and MT-II promoters was performed by introducing deletion mutants of the 5'-flanking regions into vector pGL-3, directly upstream of the firefly luciferase reporter gene. Each construct was tested in the HepG2 cell lines in the absence or presence of zinc or cadmium ions. Maximum inducibility of the MT-II gene promoter was achieved with a construct containing both the proximal and the distal MRE clusters. The lack of the most distally located MRE dramatically affected MT-II promoter sensitivity to metals; removal of the distal cluster of MREs also reduced metal inducibility. The MT-I promoter was more compact, since maximal activity and metal inducibility depended on the presence of the proximal cluster of four MREs. This study suggests that the different organization of the MT-I and MT-II gene promoter regions might account for the observed differences in the basal and metal-induced expression of MT-I and MT-II isoforms in the C. hamatus liver.

Serum lipids in humans fed diets containing beef or fish and poultry.

One half of a group of 129 men and women (74 men and 55 women), in a cross-over design at, within a self-selected diet, one egg and at least 5 oz of beef daily for 3 months while the other half at one egg and at least 5 oz of poultry and fish daily. Then they reversed their diets for 3 months. Blood samples were drawn by venipuncture before the study started and at the end of the 3 and 6 months, for analyses of serum total cholesterol, triglycerides, and high density lipoprotein cholesterol. No statistically significant changes were found in serum lipids in men. In women serum triglycerides but not other serum lipids were significantly higher when poultry and fish had been ingested.

Seasonal-dependent effect of temperature on the response of adenylate cyclase to FSH stimulation in the oviparous lizard, Podarcis sicula.

The study of environmental factors affecting vertebrate reproduction has long interested both developmental and evolutionary biologists. Although photoperiod has been considered to be an important environmental parameter for vertebrates such as birds, temperature is probably a primary external factor responsible for reproductive cyclicity in reptiles. In spite of the progress made in the understanding of reptilian reproductive strategies and adaptations, much remains to be learned about the interplay between endocrine physiological factors, such as hormones, and environmental parameters. In this report, we have examined the effects of in vivo administered FSH on oocyte recruitment during the most significant periods of the reproductive cycle of the lizard, Podarcis sicula. The results show that when FSH is administered in proximity to the reproductive period, it stimulates oocyte growth and ovulation; when the hormone is administered at the beginning of the winter stasis it affects ovarian activity without inducing ovulation. Ovarian adenylate cyclase activity is moderately sensitive to in vitro FSH stimulation during the pre- and post-reproductive periods. The sensitivity to hormone stimulation increases significantly during the reproductive period and winter stasis. We have also tested the hypothesis that environmental temperature affects the responsiveness of ovarian adenylate cyclase to FSH stimulation. For such a purpose, we exposed animals to 28 degrees C or 4 degrees C in different periods of the ovarian cycle. The results show that, whenever the temperature applied mimics the thermal regime of the coming season, adenylate cyclase sensitivity to FSH shifts towards levels that anticipate the natural responsiveness.

Identification of a high-molecular-weight cadmium-binding protein in copper-resistant Bacillus acidocaldarius cells.

Bacillus acidocaldarius grown in the presence of Cu++ was capable of accumulating the metal in the form of a protease-sensitive high molecular weight (HMW) moiety whose formation was inhibited by actinomycin D. Only cells preadapted in Cu++ were able to grow in a Cd(++)-containing medium. A cell-free extract from cadmium-stressed cells was fractionated by gel-permeation chromatography. The majority of cadmium was found associated with a HMW protein fraction which was further purified by anion exchange chromatography and high-performance liquid chromatography. The molecular weight of the purified protein was estimated to be 23,000 by SDS-PAGE. Amino acid analysis showed a low cysteine content and an abundance of aspartate and glutamate. It is likely that the cadmium-binding protein is an essential component of the mechanism mediating recovery from heavy metal toxicity.

Pitch comparisons of acoustically and electrically evoked auditory sensations.

Cochlear implant users with some residual hearing in the non-implanted ear compared the pitch sensations produced by acoustic pure tones and pulsatile electric stimuli. Pitch comparisons were obtained for pure tones and electrical stimuli presented at different positions (electrodes) in the scala tympani, keeping the electric pulse rate fixed at 100, 250, or 800 pps. Similarly, pitch comparisons were obtained for electrical stimuli with variable pulse rates presented to two fixed electrode positions (apical and basal) in the cochlea. Both electrode position and pulse rate influenced the perceived pitch of the electrical signal and 'matched' electric and acoustic signals were found over a wide range of frequencies. There was a large variation between listeners. For some stimuli, listeners had difficulty in deciding whether the acoustic or electric stimulus was higher in pitch. Despite the variability, consistent trends were obtained from the data: higher frequencies tended to be matched by more basal electrodes for all pulse rates. Higher frequencies tended to be matched by higher pulse rates for both electrode positions. The electrode positions that 'matched' pure tones were more basal than predicted from the characteristic frequency coordinates of the basilar membrane in a normal human cochlea.

Isolation and primary structure determination of a metallothionein from Paracentrotus lividus (Echinodermata, Echinoidea).

A low-molecular-mass zinc-binding protein was purified from the eggs of the sea urchin Paracentrotus lividus using procedures that included gel-permeation and anion-exchange chromatography followed by HPLC. The primary structure of this protein was derived from the sequences of peptide fragments obtained by digestion with trypsin and thermolysin. The reconstructed sequence showed the presence of 20 cysteinyl residues, thus resembling that of a metallothionein. The Paracentrotus protein was most similar to the metallothionein of Strongylocentrotus purpuratus, another member of the order of Echinoida, living along the coast of the Pacific Ocean. However, the presence of non-conservative amino acid substitution, together with a deletion of two residues in the Strongylocentrotus metallothionein, make the similarity scores of the two sea urchin proteins lower than that of metallothioneins from vertebrates of the same order. In addition, the present data show that sea urchin metallothioneins display no homology with metallothioneins of any other species.

[Importance of dermatologic screening within the frame work of a general cancer prevention program]. / Importanza di uno screening dermatologico nell'ambito di un programma di prevenzione oncolologica generale.

BACKGROUND: Skin tumours represent about 11% of all the malignant neoplasms and their frequency is increasing annually. Skin tumours (melanoma, basal and squamous cell carcinoma, etc.) can be used for a good screening activity, but in relation to breast or cervix uteri cancer needs to be better defined. A test on a population of selected patients against skin malignant neoplasms has been carried out in our Centre. All of them had skin lesions and further checks were necessary. METHODS: The diagnostic protocol used in our Centre for Oncological Prevention uses the collection of anamnestic data and an objective examination. Between 1996 and 2000, 222 patients between the ages of 18 and 80 have been selected. All of them had suspected skin lesions. The patients were selected by the oncologist, particularly for pigmentation, asymmetry, irregular borders and heterogeneous colour of their skin lesions. Subsequently, the patients were sent for a further examination to the dermatologist oncologist, who on the basis of the objective dermatological examination with possible dermatoscopy, made a clinical diagnosis of the skin injuries or suggested surgical removal for the histological control of the same. RESULTS: Requested consultations: 222. Exami-nations made: 195. Patients considered: 190. Skin injuries examined: 190. The following skin lesions were identified: melanoma: 4 (2.1%) [2: I Clark level; 2: II Clark level]; basal cell carcinoma: 14 (7.37%); dermatofibrosarcoma: 1 (0.53%); keratoacanthoma: 1 (0.53%); dysplastic nevus: 4 (2.1%); actinic keratosis: 7 (3.68%); benign lesions: 159 (83.68%). CONCLUSIONS: These data were obtained by a screening program and it is therefore not a random study. This study shows interesting results because tumoral skin lesions and in particular melanoma were recognised at early stages. This is more than enough for us to create a specific screening program for skin lesions to cut down the rate of morbidity and mortality.

[Influence of the vitamin D receptor gene polymorphism and 25-hydroxyvitamin D on arterial pressure in health individuals]. / Influencia del polimorfismo del gen receptor de la vitamina D y de la 25-hidroxivitamina D en la tensión arterial de individuos sanos.

UNLABELLED: The role of vitamin D in the regulation of blood pressure is unclear. There are no studies that relate Bsm I polymorphism with blood pressure. OBJECTIVE: To analyze if Bsm I polymorphism and 25-hydroxyvitamin D (25OHD3) influence blood pressure in healthy individuals with normal blood pressure. METHODS: Systolic (SBP) and diastolic (DBP) blood pressure, Body Mass Index (BMI), plasma creatinine, serum calcium, serum phosphorus, serum iPTH, serum 25OHD3 and Bsm I genotype were determined in 590 healthy individuals (260 men and 330 women). Data were analysed using a multiple linear regression model. SBP and DBP were defined as dependent variables and the rest of variables as independent. RESULTS: Gender was strongly associated with both SBP (beta: -12.01, p: 0.000) and DBP (beta: -4.78, p: 0.000). Therefore, a separate analysis was performed according to gender. In males, SBP was associated with BMI (beta: 0.83, p: 0.001), 25OHD3, (beta: 0.36, p: 0.000) and genotype (beta: -3.90, p: 0.002); and DBP with 25OHD 3 (beta: 0.16, p: 0.018) and age (beta: 0.28, p: 0.000). Differences of blood pressure among the three genotypes were explored by analysis of variance. SBP was higher in men with bb genotype than in the other genotypes (p: 0.007). In females, 25OHD3 and genotype were not associated with blood pressure. CONCLUSIONS: Healthy men with higher levels of vitamin D have higher levels of SBP and DBP. Moreover, men with bb genotype have the highest levels of SBP. Blood pressure levels in women are not influenced by vitamin D nor by Bsml genotype. Our data suggest a possible pathophysiological interaction between vitamin D and sex hormones in blood pressure control.

[Differential effect of vitamin D analogues on the proliferation of vascular smooth muscle cells]. / Efecto diferencial de los análogos de la vitamina D en la proliferación de células de músculo liso vascular.

Atherosclerosis is the principal cause of myocardial infarction, stroke, and peripheral vascular disease, accounting for nearly half of all mortality in developed countries. The excessive growth of vascular smooth muscle cells is an important component in the development of atherosclerotic lesion. The direct effect of calcitriol and vitamin D analogs on the VSMCs proliferation is not clear. In this study we have analysed if calcitriol, Paricalcitol (19-nor-1,25-dihydroxy-vitamin D2) and EB1089 (experimental analog used as anticancerous) modify proliferation and the expression of vitamin D receptor (VDR) gene that is regulated at the transcriptional level by itself in the VSMCs. VSMCs proliferation was analysed by BrdU incorporation and VDR gene expression using RT-PCR. VSMCs proliferation was stimulated when calcitriol was added to the culture. VSMCs proliferation was significantly lower with analogs at the same dose. With regard to the functional study, the expression of VDR gene was upregulated by calcitriol at a concentration of 100 nM. There were no changes in this expression with the analogs. In conclusion, calcitriol, do not modify VSMCs proliferation. Therefore, Paricalcitol could have a minor proliferating effect on the wall of vessels that vitamin D.