Mapping of α- and β-globin genes on Antarctic fish chromosomes by fluorescence in-situ hybridization

The pathways and mechanisms of genomic change that have led to the peculiar haemoglobinless phenotype of the white-blooded Antarctic icefishes (16 species in the family Channichthyidae) constitute an important model for understanding the rapid diversification of the Antarctic notothenioid fish flock. To provide complementary structural information on genomic change at globin-gene loci in Antarctic fish species, cytogenetic studies and in-situ chromosomal mapping have been undertaken. Using a DNA probe containing one α- and one β-globin gene from the embryonic/juvenile globin gene cluster of the red-blooded species Notothenia coriiceps, we mapped the cluster on the chromosomes of Antarctic teleosts by fluorescence in-situ hybridization. As anticipated on the basis of its molecular organization, the cluster was located on a single chromosome pair in all of the red-blooded fish species probed (N. coriiceps, N. angustata, Trematomus hansoni, T. pennellii). In contrast, the α/β-globin probe did not recognize complementary sequences on the chromosomes of the white-blooded species Chionodraco hamatus and Channichthys rhinoceratus. These results represent the first example of chromosomal mapping of embryonic/juvenile globin genes in teleostean fishes. Beyond its relevance to the evolutionary history of Antarctic notothenioids, this work contributes to our understanding of the evolution of the chromosomal loci of globin genes in fishes and other vertebrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Systemic absorption of 3H-fenticonazole after vaginal administration of 1 gram in patients

Fourteen women, five with normal cervicovaginal mucosa (Group 1), five with cervical carcinoma (Group 2) and four with relapsing vulvovaginal candidiasis (Group 3) were enrolled and completed this open clinical trial. Each subject received a single dose of 1.82 +/- 0.3 g on average of vaginal paste (for ovules) containing about 1000 mg of 3H-fenticonazole nitrate (266 microCi). Twelve hours after vaginal administration, the paste was removed by vaginal washing. Blood, urine and stool samples were collected at specified time intervals for five days. Plasma, urine, stools and all used material in contact with the paste were assayed for radioactivity. No measurable levels of radioactivity were detected in plasma of subjects of Groups 1 and 3 while in 4 of the 5 subjects with cervical carcinoma (Group 2) fenticonazole was detected during the 24 h after administration with a peak level at about 8 hours. For a period of 5 days, 0.4-1.5% of the dose on average was recovered from urine, and 0.18-0.32% from feces. Based on the excretion data, the extent of vaginal absorption of fenticonazole nitrate in women with vulvovaginal candidiasis was 1.81 +/- 0.57% of the dose, while in women with normal cervicovaginal mucosa it accounted for 0.58 +/- 0.28% of the administered dose. In patients with cervical carcinoma, absorption was 1.12 +/- 0.53%. The maximum amount absorbed corresponds to an exposure of about 0.4 mg/kg of fenticonazole nitrate (for a subject weighing 50 kg). Consequently, the vaginal administration of one ovule containing 1000 mg of fenticonazole nitrate seems to be devoid of risk for patients.     

Similar CD40 ligand expression on EL-4 thymoma cell lines with widely different helper activity for B lymphocytes.

A mutagenized subclone of the murine EL-4 thymoma (clone B5) is approximately 30 times more potent than parental EL-4 cells in stimulating proliferation and Ig secretion of murine or human B cells by direct cell contact in the presence of appropriate cytokines. In this study we found that CD40 ligand (CD40L) expression was constitutive and very similar on EL-4 B5 and parental EL-4 cells according to Northern blot and flow cytometry. Activation with phorbol 12-myristate 13-acetate (PMA) alone, PMA and ionomycin, interleukin-1 (IL-1) or human T-cell supernatant did not lead to significant CD40L up-regulation. A receptor-binding assay with soluble CD40 did not reveal different ligand affinities. However, murine and human soluble CD40-IgFc fusion proteins inhibited human B-cell stimulation by EL-4 B5 cells in the presence of human T-cell supernatant. Inhibition was 96% when soluble CD40 was added on day 0 of culture and progressively decreased when the CD40 was added subsequently. Ig secretion by cytoplasmic Ig-positive cells was no longer inhibited. These findings imply that, although CD40 ligand is necessary for B-cell activation by EL-4B5 cells, additional molecule(s) must be responsible for the increased helper activity of the EL-4 B5 clone.     

Could mitochondrial haplogroups play a role in sporadic amyotrophic lateral sclerosis?

Mitochondrial impairment has been implicated in the pathogenesis of the amyotrophic lateral sclerosis (ALS). Furthermore, mitochondrial-specific polymorphisms were previously related to other neurodegenerative diseases, such as Parkinson, Friedreich and Alzheimer disease. To investigate if specific genetic polymorphisms within the mitochondrial genome (mtDNA) could act as susceptibility factors and contribute to the clinical expression of sporadic ALS (sALS), we have genotyped predefined European mtDNA haplogroups in 222 Italian patients with sALS and 151 matched controls. Individuals classified as haplogroup I demonstrated a significant decrease in risk of ALS versus individuals carrying the most common haplogroup, H (odds ratio 0.08, 95% confidence interval 0.04-0.4, p < 0.01). Further stratification of the dataset by sex, age and site of onset of disease and survival failed to reach significance for association. Our study provides evidence of the contribution of mitochondrial variation to the risk of ALS development in Caucasians. Further it may help elucidate the mechanism of the mitochondrial dysfunction detectable in ALS, and may be of relevance in development of strategies for the treatment of this disease.     

N,N-dialkylaminosubstituted chromones and isoxazoles as potential anti-inflammatory agents.

The ability of some N,N-dialkylaminosubstituted chromones and isoxazoles to inhibit the protein kinase C (PKC) dependent signal transduction pathway was tested. As a cellular model, human neutrophils stimulated with either phorbol myristate acetate (PMA) or formylmethionine-leucine-phenylalanine (f-MLF) were used. The efficiency of the compounds was established by their capacity to reduce the O2- production by activated human neutrophils. Compounds carrying a 3-bis(2-methoxyethyl)amino group, a substituent found active in previously tested tricyclic compounds, do not show significant anti-PKC activity in this study. On the other hand, substitution with a 1-piperidinyl group leads all tested compounds to a high biological activity against stimulated neutrophils.     

Posterior knee pain: primary symptom of a small non-occlusive venous clot.

A healthy 9 year old girl presented with severe posterior knee pain and a small segmental non-occlusive popliteal venous thrombosis. The case is relevant for its unique presentation and symptoms. Lack of recanalisation persisted at one year follow up.     

Radiation damage to triplex DNA induced by gamma-rays: a footprinting study and Monte Carlo simulation

PURPOSE: This study is aimed at comparing the radiosensitivity of a 21-mer DNA fragment in duplex and triplex form (Py x Pu duplex and Py x Pu:Py triplex) and to give insights into the fine structural features due to the different strandedness. MATERIALS AND METHODS: The triplex sample was characterized by means of electrophoretic mobility, circular dichroism spectra and UV melting experiments. Triplex and duplex, labelled on the homopyrimidine or homopurine strand, were irradiated with 60Co gamma-rays. The samples were analysed by sequencing gel electrophoresis and the patterns of relative probabilities of frank strand breakage (FSB) and alkali-revealed breakage (ARB) at each nucleotide site were determined. Relative probabilities of OH* radical attack to duplex and triplex DNA built up with a molecular modelling software were calculated using a previously reported simulation procedure (Sy et al. 1997). The experimental data were compared with the results of the theoretical simulations of OH* radical attack to DNA. RESULTS: Globally, the duplex is more attacked than the triplex, mainly in the Pu strand. Sequence-dependent variations of FSB and FSB+ARB probabilities in both duplex and triplex were revealed. No significant differences between the patterns of damage in the triplex and in the duplex were observed. CONCLUSIONS: The presence of the third Py strand located in the major groove of the DNA duplex modifies the total yields of radiation-induced DNA damage, but not the sequence-dependent patterns of relative probability of damage at each nucleotide site.     

Fiberoptic bronchoscopy in the diagnosis of pulmonary lymphomas

Fiberoptic bronchoscopy was useful for the diagnosis of 7 of 9 pulmonary lymphomas (5 primary lung lymphomas, 4 lung involvement in systemic disease). Radiologic and endoscopic findings were variable. The importance of immunohistochemical markers for successful diagnosis is emphasized.     

A common mutation of the insulin receptor substrate-1 gene is a risk factor for coronary artery disease

Insulin resistance is associated with increased risk of atherosclerosis. Insulin receptor substrate-1 (IRS-1) plays a key role in tissue insulin sensitivity. A common mutation (G972R) of the IRS-1 gene has been shown to impair IRS-1 function, and it has been associated with reduced insulin sensitivity and lipid abnormalities. This led us to investigate the role of the G972R mutation in predisposing individuals to coronary artery disease (CAD). The DNA of 318 subjects with angiographically documented coronary atherosclerosis (>50% stenosis) and 208 population control subjects was analyzed for the presence of the G972R mutation. This mutation was detected by nested polymerase chain reaction and BstNI restriction enzyme digestion. The frequency of the G972R mutation was significantly higher among patients with CAD than controls (18. 9% versus 6.8%, respectively; P<0.001). After controlling for other coronary risk factors, the relative risk of CAD associated with the G972R mutation was 2.93 (95% CI 1.30 to 6.60; P<0.02) in the entire cohort. This risk was found to be even higher in the subgroups of obese subjects (odds ratio [OR] 6.97, 95% CI 2.24 to 21.4; P<0.001) and subjects with clinical features of insulin resistance syndrome (OR 27.3, 95% CI 7.19 to 104.0; P<0.001). The IRS-1 gene variant was associated with a higher frequency of diabetes mellitus (14.9% among carriers versus 6.5% among noncarriers; P<0.01) and with a 60% increase of plasma total triglycerides (P<0.001). Also, plasma concentrations of total cholesterol and the ratio of total cholesterol to HDL cholesterol were significantly (P<0.001) higher among carriers than noncarriers, although to lesser a extent. These effects were independent of CAD status. The G972R mutation in the IRS-1 gene was found to be a significant independent predictor of CAD. Moreover, this mutation greatly increased the risk of CAD in obese subjects and in patients with the cluster of abnormalities of insulin resistance syndrome. Besides the increased frequency of diabetes, carriers showed a more atherogenic lipid profile, suggesting a potential role of the IRS-1 gene in the pathogenesis of lipid abnormalities associated with CAD.     

Amyotrophic lateral sclerosis: a new missense mutation in the SOD1 gene

Copper-zinc superoxide dismutase-1 (SOD1) is the second most common mutated gene in amyotrophic lateral sclerosis (ALS). To date more than 150 missense mutations of SOD1 have been reported. The objective of this study was to describe a novel SOD1 mutation and its phenotypic expression. We describe a 74-year-old Caucasian man who began to complain of progressive weakness and atrophy of the right hand and over 10 months developed a severe tetraparesis, with atrophies of upper and lower limbs and neck muscles, dysphagia, and dyspnea that led to percutaneous endoscopic gastrostomy and tracheotomy. A diagnosis of ALS was made. Genetic analysis identified a heterozygous mutation in exon 4 of SOD1 that results in the amino acid substitution from arginine to cysteine at position 115 (p.R115C). We identified a novel pathogenic SOD1 mutation in a patient with a very rapid disease progression and aggressive phenotype providing additional information on the wide range of SOD1 mutations in apparently sporadic ALS and confirming the possibility of a strong genotype-phenotype correlation for distinct SOD1 mutations.