Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

Symbols are not uniquely human

Modern semiotics is a branch of logics that formally defines symbol-based communication. In recent years, the semiotic classification of signs has been invoked to support the notion that symbols are uniquely human. Here we show that alarm-calls such as those used by African vervet monkeys (Cercopithecus aethiops), logically satisfy the semiotic definition of symbol. We also show that the acquisition of vocal symbols in vervet monkeys can be successfully simulated by a computer program based on minimal semiotic and neurobiological constraints. The simulations indicate that learning depends on the tutor-predator ratio, and that apprentice-generated auditory mistakes in vocal symbol interpretation have little effect on the learning rates of apprentices (up to 80% of mistakes are tolerated). In contrast, just 10% of apprentice-generated visual mistakes in predator identification will prevent any vocal symbol to be correctly associated with a predator call in a stable manner. Tutor unreliability was also deleterious to vocal symbol learning: a mere 5% of "lying" tutors were able to completely disrupt symbol learning, invariably leading to the acquisition of incorrect associations by apprentices. Our investigation corroborates the existence of vocal symbols in a non-human species, and indicates that symbolic competence emerges spontaneously from classical associative learning mechanisms when the conditioned stimuli are self-generated, arbitrary and socially efficacious. We propose that more exclusive properties of human language, such as syntax, may derive from the evolution of higher-order domains for neural association, more removed from both the sensory input and the motor output, able to support the gradual complexification of grammatical categories into syntax.     

A method for 3D detection of symmetry line in asymmetric postures

The methods for symmetry line detection presented in the literature are typically suited to analyse symmetric upright postures, both standing and seated. The proposed method focuses on the symmetry line detection in subjects assuming asymmetric postures in which this line falls far outside the sagittal plane. The proposed approach evaluates the symmetry line by means of an autoregressive process in order to determine the set of planes suited to slice the back coherently with its geometric spatial configuration. The method is analysed assuming the cutaneous marking as reference and it is compared with a previous one, also developed by these authors. Results are analysed and critically discussed.     

VEGF,HIF-1α Expression and MVD as an Angiogenic Network in Familial Breast Cancer

Angiogenesis, which plays an important role in tumor growth and progression of breast cancer, is regulated by a balance between pro- and anti-angiogenic factors. Expression of vascular endothelial growth factor (VEGF) is up-regulated during hypoxia by hypoxia-inducible factor-1α (HIF-1α). It is known that there is an interaction between HIF-1α and BRCA1 carrier cancers, but little has been reported about angiogenesis in BRCA1-2 carrier and BRCAX breast cancers. In this study, we investigated the expression of VEGF and HIF-1α and microvessel density (MVD) in 26 BRCA1-2 carriers and 58 BRCAX compared to 77 sporadic breast cancers, by immunohistochemistry. VEGF expression in BRCA1-2 carriers was higher than in BRCAX cancer tissues (p = 0.0001). Furthermore, VEGF expression was higher in both BRCA1-2 carriers and BRCAX than the sporadic group (p<0.0001). VEGF immunoreactivity was correlated with poor tumor grade (p = 0.0074), hormone receptors negativity (p = 0.0206, p = 0.0002 respectively), and MIB-1-labeling index (p = 0.0044) in familial cancers (BRCA1-2 and BRCAX). The percentage of nuclear HIF-1α expression was higher in the BRCA1-2 carriers than in BRCAX cancers (p<0.05), and in all familial than in sporadic tumor tissues (p = 0.0045). A higher MVD was observed in BRCA1-2 carrier than in BRCAX and sporadic cancer tissues (p = 0.002, p = 0.0001 respectively), and in all familial tumors than in sporadic tumors (p = 0.01). MVD was positively related to HIF-1α expression in BRCA1-2 carriers (r = 0.521, p = 0.006), and, in particular, we observed a highly significant correlation in the familial group (r = 0.421, p<0.0001). Our findings suggest that angiogenesis plays a crucial role in BRCA1-2 carrier breast cancers. Prospective studies in larger BRCA1-2 carrier series are needed to improve the best therapeutic strategies for this subgroup of breast cancer patients.     

Opposite Roles of NMDA Receptors in Relapsing and Primary Progressive Multiple Sclerosis

Synaptic transmission and plasticity mediated by NMDA receptors (NMDARs) could modulate the severity of multiple sclerosis (MS). Here the role of NMDARs in MS was first explored in 691 subjects carrying specific allelic variants of the NR1 subunit gene or of the NR2B subunit gene of this glutamate receptor. The analysis was replicated for significant SNPs in an independent sample of 1548 MS subjects. The C allele of rs4880213 was found to be associated with reduced NMDAR-mediated cortical excitability, and with increased probability of having more disability than the CT/TT MS subjects. MS severity was higher in the CC group among relapsing-remitting MS (RR-MS) patients, while primary progressive MS (PP-MS) subjects homozygous for the T allele had more pronounced clinical worsening. Mean time to first relapse, but not to an active MRI scan, was lower in the CC group of RR-MS patients, and the number of subjects with two or more clinical relapses in the first two years of the disease was higher in CC compared to CT/TT group. Furthermore, the percentage of relapses associated with residual disability was lower in subjects carrying the T allele. Lesion load at the MRI was conversely unaffected by the C or T allele of this SNP in RR-MS patients. Axonal and neuronal degeneration at the optical coherence tomography was more severe in the TT group of PP-MS patients, while reduced retinal nerve fiber thickness had less consequences on visual acuity in RR-MS patients bearing the T allele. Finally, the T allele was associated with preserved cognitive abilities at the Rao’s brief repeatable neuropsychological battery in RR-MS. Signaling through glutamate NMDARs enhances both compensatory synaptic plasticity and excitotoxic neurodegeneration, impacting in opposite ways on RR-MS and PP-MS pathophysiological mechanisms.     

Molecular, Serological, and Virulence Characteristics of Vibrio parahaemolyticus Isolated from Environmental, Food, and Clinical Sources in North America and Asia

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.     

Vgamma9/Vdelta2 T lymphocytes in Italian patients with Behçet's disease: evidence for expansion,and tumour necrosis factor receptor II and interleukin-12 receptor beta1 expression in active disease

Beh?et's disease is a multisystem disease in which there is evidence of immunological dysregulation. It has been proposed that gamma/delta T cells are involved in its pathogenesis. The aim of the present study was to assess the capacity of gamma/delta T cells with phenotype Vgamma9/Vdelta2, from a group of Italian patients with Beh?et's disease, to proliferate in the presence of various phosphoantigens and to express tumour necrosis factor (TNF) and IL-12 receptors. Twenty-five patients and 45 healthy individuals were studied. Vgamma9/Vdelta2 T cells were analyzed by fluorescence activated cell sorting, utilizing specific monoclonal antibodies. For the expansion of Vgamma9/Vdelta2 T cells, lymphocytes were cultured in the presence of various phosphoantigens. The expression of TNF receptor II and IL-12 receptor beta1 was evaluated with the simultaneous use of anti-TNF receptor II phycoerythrin-labelled (PE) or anti-IL-12 receptor beta1 PE and anti-Vdelta2 T-cell receptor fluorescein isothiocyanate. There was a certain hierarchy in the response of Vgamma9/Vdelta2 T cells toward the different phosphoantigens, with the highest expansion factor obtained with dimethylallyl pyrophosphate and the lowest with xylose 1P. The expansion factor was fivefold greater in patients with active disease than in those with inactive disease or in control individuals. TNF receptor II and IL-12 receptor beta1 expressions were increased in both patients and control individuals. The proportion of Vgamma9/Vdelta2 T cells bearing these receptors was raised in active disease when Vgamma9/Vdelta2 T cells were cultured in the presence of dimethylallyl pyrophosphate. These results indicate that Vgamma9/Vdelta2 T cell activation is correlated with disease progression and probably involved in the pathogenesis.     

A consensus linkage map of barley

A consensus linkage map of the barley genome was constructed. The map is based on six doubled haploid and one F₂ population. The mapping data for three of the doubled haploid populations was obtained via the GrainGenes database. To allow merger of the maps, only RFLP markers that produce a single scorable band were included. Although this reduced the available markers by about half, the resultant map contains a total of 587 markers including 87 of known function. As expected, gene order was highly conserved between maps and all but two discrepancies were found in closely linked markers and are likely to result from the small population sizes used for some maps. The consensus map allows the rapid localisation of markers between published maps and should facilitate the selection of markers for high-density mapping in defined regions.