Cloning and functional characterization of the 5''-flanking region of human methionine adenosyltransferase 2A gene

Electron microscopy of dimeric and trimeric single chain antibody Fv fragments (scFvs) complexed with anti-idiotype Fab fragments was used to reveal the orientation of antigen binding sites. This is the first structural analysis that discloses the multivalent binding orientation of scFv trimers (triabodies). Three different scFv molecules were used for the imaging analysis; NC10 scFv-5 and scFv-0, with five- and zero-residue linkers respectively between the VH and VL domains, were complexed with 3-2G12 anti-idiotype Fab fragments and 11-1G10 scFv-0 was complexed with NC41 anti-idiotype Fab fragments. The scFv-5 molecules formed bivalent dimers (diabodies) and the zero-linker scFv-0 molecules formed trivalent trimers (triabodies). The images of the NC10 diabody-Fab complex appear as boomerangs, not as a linear molecule, with a variable angle between the two Fab arms and the triabody-Fab complexes appear as tripods.     

Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features

Missense mutations in p53 frequently occur at 'hotspot' amino acids which are highly conserved and represent regions of structural or functional importance. Using the p53 mutation database and the p53 DNA sequences for 11 species, we more precisely defined the relationships among conservation, mutation frequency and protein structure. We aligned the p53 sequences codon-by-codon and determined the degree of substitution among them. As a whole, p53 is evolving at an average rate for a mammalian protein-coding gene. As expected, the DNA binding domain is evolving more slowly than the carboxy and amino termini. A detailed map of evolutionary conservation shows that within the DNA binding domain there are repeating peaks and valleys of higher and lower evolutionary constraint. Mutation hotspots were identified by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Seventy-three hotspots were identified; these 19% of codons account for 88% of all reported p53 mutations. Both high evolutionary constraint and mutation hotspots are noted at amino acids close to the protein-DNA interface and at others more distant from DNA, often buried within the core of the folded protein but sometimes on its surface. The results indicate that targeting highly conserved regions for mutational and functional analysis may be efficient strategies for the study of cancer-related genes.     

Cloning and characterization of the 5'' flanking sequences (promoter region) of the human GLP-1 receptor gene

We have investigated in rat brain slices the effects of the volatile anaesthetics enflurane, isoflurane and halothane on spontaneous discharge patterns and mean firing rates of cerebellar Purkinje cells. In the absence of these anaesthetics, Purkinje cells fired bursts of action potentials separated by quiescent periods lasting less than 2 s. Mean discharge rates were 10.8 (SEM 0.4) Hz at 23 +/- 1 degrees C and 25.6 (1.2) Hz at 35 +/- 1 degrees C. The agents exhibited qualitatively different effects when applied at concentrations corresponding to 1-3 MAC. Enflurane markedly lengthened burst and inter-burst durations. Isoflurane acted in a similar manner, but effects were less pronounced. In contrast with isoflurane and enflurane, halothane shortened burst durations. At concentrations corresponding to 1-1.5 MAC, halothane, isoflurane and enflurane significantly depressed action potential firing by 15-30% (P < 0.05). Enflurane 1.2 mmol litre-1 (2.0 MAC), isoflurane 0.9 mmol litre-1 (2.8 MAC) and halothane 0.9 mmol litre-1 (3.8 MAC) depressed spontaneous spike rates by 50%. The changes in discharge patterns and the concentration-dependent decrease in the firing rates were similar at 23 +/- 1 degrees C and 35 +/- 1 degrees C. In summary, we observed that neither the anaesthetic-induced alterations in spontaneous discharge patterns nor the EC50 values of the concentration-dependent depression of the mean firing rates were in accordance with the Meyer-Overton rule. However, at clinically relevant concentrations, depression of average spike rates did not differ significantly between the anaesthetics and thus followed the rule. Our results suggest that anaesthetic actions, which are in accordance with the rule, are frequently masked by several side effects.     

Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene

Several recent studies have reported that there is an imbalance of increased oxidant status and decreased antioxidant system in women with preeclampsia and that factor might contribute to the pathogenesis of the disease. The following study examined blood levels of some of the antioxidants (glutathione, glutathione-peroxidase and -SH groups) as markers of lipid peroxidation in women with preeclampsia compared with normal gestation. Blood levels of these antioxidants were found significantly decreased in women with preeclampsia, which allowed us to speculate that there were abnormally increased levels of lipid peroxides. We believe that lipid peroxides are toxic compounds that damage endothelial cells, increase peripheral vasoconstriction and increase thromboxane synthesis and decrease prostacyclin synthesis. We consider that lipid peroxides are not the cause but the effect of oxidative stress induced by ischaemic placenta and leukocytes activation, so the contribute but not induce pathogenesis in preeclampsia.     

Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.     

Cloning and characterization of the 5'-flanking region of the human cardiotrophin-1 gene

To enable the analysis of the regulation of the human cardiotrophin-1 gene expression, the 5'-flanking region of the human cardiotrophin-1 gene was cloned and sequenced. Data bank search revealed several cis- active DNA elements (SP1, CREB, C/EBP, AP1 and AP-2 like and GATA) in the proximal 1.1 kb region. Six nested 5-'terminal deletion mutants from -1091/+39 to -218/+39 were fused to a luciferase reportergene and proved to be functionally active after transfection into COS-7 cells.     

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.     

p53 gene mutation: software and database

Facial aging is a biological phenomenon. Skin properties change with time, and gravity and facial expressions exert mechanical deformation. Knowledge of these alterations may suggest ways to reverse them by identifying the corresponding distortional forces. The aim of this study was to determine a pattern of change for parameters of the face during the aging process, based on the numerical fitting of measures from a sample of patients. The first aspect of this study was to define adequate facial parameters and means of measuring them. Subsequently, each parameter was defined individually, and these data were analyzed as a set. The sample for the research was restricted to a group of 40 white female patients with a history of limited exposure to the sun, with ages ranging from 25 to 65. The reason for choosing this sample was the availability of frontal pattern photographs at different ages. The parameters for each patient were measured at two different ages. A strong correlation was found between age and behavior of the parameters. This aging model can be verified qualitatively by comparing photographs of a patient with manipulated photographs simulating aging. The quantitative verification of the model was done through the comparison of the measured and the predicted parameters.