Sex determinants in the genome--lessons from the animal kingdom

The immense value of sex differentiation as a means of enriching and evolving the genome has been proven by the vast variety of sex determining mechanisms to which organisms of all kinds resort. From single gene switching pathways found in lower level organisms to haplodiploid reproduction in hymenoptera, temperature-determined sex in reptiles and sex chromosomes in mammals and avians, nature and evolution have designated an impressive amount of effort to ensure that sex-specific variations remain under well-regulated control. Therefore enhancing our efforts to study some of the strategies recruited for the above may also lead to a better understanding of the inherent complexity of sexual dimorphism in general.     

Real-time detection of BRCA1 gene mutations using a monolithic silicon optocoupler array

The monolithic silicon optocoupler presented here offers a platform for a new generation of fully integrated devices fabricated through the mature and inexpensive silicon technology. Using the developed optocoupler, real-time detection of the most common mutations in BRCA1 gene related to predisposition to hereditary breast/ovarian cancer was accomplished. For this purpose, oligonucleotides corresponding to wild- and mutant-type sequences were immobilized onto different optocouplers and the hybridization with fluorescently labeled complementary or non-complementary sequences was monitored in real time. Hybridization of fluorescently labeled oligonucleotides to the immobilized ones modulated the coupling efficiency between the light emitting diode and the detector in a concentration-dependent manner. Using as label the AlexaFluor 647 dye (whose absorption maximum fits the emission maximum of the light source) a detection limit of 0.9 nM (9 fmol) was achieved. Real-time signal monitoring, especially during dehybridization, improved considerably the discrimination between wild-type and mutant sequences due to the ability to calculate dissociation kinetics upon washing independently for each one mutation. The bioanalytical capabilities of the transducer along with the fact that dense transducer arrays can be fabricated on a single chip open new frontiers in the manufacturing of microsystems appropriate for point-of-care analysis.     

Synthesis and biological evaluation of benzopyran analogues bearing class III antiarrhythmic pharmacophores

We have synthesized a series of compounds combining the hydroxy-benzopyran ring of vitamin E with the methylsulfonylaminophenyl group of class III antiarrhythmic drugs, connected through tertiary amine moieties. Evaluation of the antiarrhythmic and antioxidant activity of the new compounds was carried out on isolated rat heart preparations using the non-recirculating Langendorff mode. The new analogues were present, at 10 microM concentration, during ischemia and reperfusion. Selected compounds were further studied by a conventional microelectrode method in order to get insight into their cellular mode of action. The most active compound, N-[4-[2-[[2-(3,4-dihydro-6-hydroxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5-yl)ethyl] methylamine]ethyl]phenyl]methanesulfonamide (19a), reduces premature beats, prolongs QT and QRS intervals during ischemia and reperfusion, and reduces MDA content, leading to a fast recovery of the heart. In addition, it exhibits moderate class III antiarrhythmic action.     

Chronic exposure to low levels of antigen in the periphery causes reversible functional impairment correlating with changes in CD5 levels in monoclonal CD8 T cells

This study describes a double-transgenic model in which monoclonal CD8 F5 T cells are chronically exposed to self Ag (nucleoprotein) in the periphery, but are not affected during thymic development. Chronic exposure of CD8 T cells to their cognate Ag rendered them unable to proliferate or produce cytokines in response to antigenic stimulation in vitro. However, the cells still retained some killer function in vivo and continuously eliminated APC expressing high levels of Ag. In addition, when crossed with mice expressing Ag in the anterior pituitary gland (triple-transgenic mice), F5 T cells migrated to this site and killed growth hormone producing somatotrophs. The anergic state was reversible upon transfer into Ag-free recipients, resulting in full recovery of in vitro responsiveness to Ag. Anergic CD8 T cells express higher levels of CD5, a negative regulator of T cell signaling, whereas after transfer and residence in Ag-free hosts, CD5 levels returned to normal. This suggests that up-regulation of negative T cell regulators in peripheral T cells exposed to chronic stimulation by Ag may prevent full functionality and thus avoid overt autoreactivity.     

Effects of physical activity, body fat, and salivary cortisol on mucosal immunity in children.

This study examined relationships among physical activity, body composition, and stress- and immunity-related variables in fifth grade children (10-11 yr) in Southern Ontario. The 29 boys and 32 girls, who participated in the study, performed a 20-m shuttle run for prediction of aerobic fitness. Bioelectrical impedance was used to assess relative body fat. Standardized questionnaires were used to determine physical activity-related variables and frequency of upper respiratory tract infection (URTI). Resting saliva samples were collected and tested for resting cortisol and resting secretory immunoglobulin A (SIgA). Subjects wore a pedometer for 48 h to estimate their average total distance traveled per day. SIgA was significantly correlated with reported URTIs but was not related to salivary cortisol, physical activity, fitness level, or relative body fat. Children who spent more time in sport activities and had higher aerobic fitness reported fewer "sick" days. Children with body fat higher than 25% reported significantly (P < 0.05) more sick days than the rest of the cohort. There were no gender differences in SIgA, URTI frequency, and cortisol levels. The test-retest reproducibility for salivary cortisol was 0.66 (P < 0.01), whereas long-term SIgA reproducibility was nonsignificant for repeated measurements taken after 6 wk. Resting secretory immunity was not strongly related to fitness and physical activity, but there was evidence that reduced physical activity and excess body fat can result in higher URTI incidence.     

Hygromycin B-selected cell lines from GAL4-regulated pUAST constructs

Here we report the generation of stable, selectable Drosophila S2 cell lines using the UAS-GAL4 system. Cloning of the hygromycin resistance gene into the pUAST vector and cotransfection with other pUAST constructs in S2 cells results in coexpression of up to four different proteins under hygromycin selection. Protein expression is driven by the ubiquitous Actin5C-GAL4 driver and cell cultures are maintained in hygromycin-supplemented, serum-free media to ensure constitutive protein production. Visual comparison of cells cotransfected with GFP and RFP demonstrates a uniform cell population expressing both markers simultaneously, while Western blot analysis shows concurrent expression of MYC3-tagged proteins. In addition, fluorescent cell sorting (FACS) analysis shows that 80% of the total cell population express the GFP marker. Our data indicate that using this technique it is possible to establish stable, selectable cell lines that provide a pool of readily accessible protein. This facilitates protein-based studies and abolishes the need to carry out time-consuming and expensive transfections.     

Bifunctional agents for reperfusion arrhythmias: novel hybrid vitamin E/class I antiarrhythmics

We have synthesized a series of hybrid compounds combining the pharmacophoric redox moieties of vitamin E and key features responsible for the antiarrhythmic properties of the class I antiarrhythmics procainamide and lidocaine. Procainamide analogue (2a) and lidocaine analogues (14a) and (14b) are very strong inhibitors of lipid peroxidation. All analogues tested at 100 or 30 microM enhanced the post ischemic recovery without inducing ventricular fibrillations while there was no evidence in our experiments for drug-induced pro-arrhythmia. In addition, they induced a widening of the QRS intervals. Our data suggest that the efficacy of the new compounds in preventing reperfusion arrhythmias could be attributed to their combined effects involving inhibition of free radical mediated damage coupled with antiarrhythmic properties.     

The effect of lower body positive pressure on the cardiovascular response to exercise in sedentary and endurance-trained persons with paraplegia

Exercise intolerance in persons with paraplegia (PARAS) is thought to be secondary to insufficient venous return and a subnormal cardiac output at a given oxygen uptake. However, these issues have not been resolved fully. This study utilized lower-body positive pressure (LBPP) as an intervention during arm crank exercise in PARAS in order to examine this issue. Endurance-trained (TP, n= 7) and untrained PARAS (UP, n= 10) with complete lesions between T6 and T12, and a control group consisting of sedentary able-bodied subjects (SAB, n= 10) were tested. UP and TP subjects demonstrated a diminished cardiac output (via CO₂ rebreathing) during exercise compared to SAB subjects. Peak oxygen uptake (V˙O_2peak) remained unchanged for all groups following LBPP. LBPP resulted in a significant decrease in heart rate (HR) in UP and TP (P≤0.05), but not SAB subjects. LBPP produced an insignificant increase in cardiac output (Q˙) and stroke volume (SV). The significant decrease in HR in both PARA groups may indicate a modest hemodynamic benefit of LBPP at higher work rates where circulatory sufficiency may be most compromised. We conclude that PARAS possess a diminished cardiac output during exercise compared to the able-bodied, and LBPP fails to ameliorate significantly their exercise response irrespective of the conditioning level. These results support previous observations of a lower cardiac output during exercise in PARAS, but indicate that lower-limb blood pooling may not be a primary limitation to arm exercise in paraplegia. Accepted: 11 December 1997     

Immunobiology of HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein and its potential application in cancer immunotherapy

HER-2/neu (also known as HER2 or c-erb-B2) is a 185-kDa protein receptor with tyrosine kinase activity and extensive homology to the epidermal growth factor (EGF) receptor. HER-2/neu is expressed in many epithelial tumors and known to be overexpressed in approximately 20–25% of all ovarian and breast cancers, 35–45% of all pancreatic adenocarcinomas, and up to 90% of colorectal carcinomas. HER-2/neu overexpression represents a marker of poor prognosis. HER-2/neu-positive tumor cells are potentially good targets for tumor-reactive cytotoxic T lymphocytes which have been utilized in immunotherapeutic trials. In addition, the humanized monoclonal antibody Herceptin has been tested in several clinical trials and proved to be an effective adjuvant therapy for HER-2/neu-positive breast and ovarian cancers. Vaccinations aiming at generating T-cell responses are being examined in both experimental and clinical trials. Natural immunity at the level of T and B cells has been observed in patients with HER-2/neu-positive tumors confirming the immunogenicity of HER-2/neu and encouraging vaccination trials with HER-2 protein–derived subunits or synthetic peptides. This review summarizes recent data from patients with various types of HER-2/neu–overexpressing cancers carrying different HLA alleles and exhibiting preexistent immunity to HER-2/neu–derived synthetic peptides. It also discusses potential advantages of the various vaccination approaches to immunotherapy targeting the HER-2/neu molecule.Keywords ImmunobiologyHER-2/neu OncoproteinCancer ImmunologyThis work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.