Comprehensive genomic analysis of the dromedary T cell receptor gamma (TRG) locus and identification of a functional TRGC5 cassette.

The emergent availability in public databases of more complete genome assemblies allows us to improve genomic data obtained by classical molecular cloning. The main goal of this study was to refine the genomic map of the dromedary TRG locus by integrating our previous genomic data with the analysis of recent genomic assemblies. We identified an additional TRGC cassette, defined as a V-J-C recombination unit, located at the 5' of the locus and made up of five TRGV genes followed by three TRGJ genes and one TRGC gene. Hence, the complete dromedary TRG locus spans about 105 Kb and consists of three in tandem TRGC cassettes delimited by AMPH and STARD3NL genes at the 5' and 3' end, respectively. An expression assay carried out on peripheral blood showed the functional competency for the dromedary TRGC5 cassette and confirmed the presence of the somatic hypermutation mechanism able to enlarge the repertoire diversity of the dromedary γδ T cells.

Comparative antioxidant activity of cultivated and wild Vaccinium species investigated by EPR, human neutrophil burst and COMET assay.

OBJECTIVES: The Vaccinium (V.) spp. berries are considered a source of antioxidants, mainly belonging to polyphenols, specifically flavonoids and anthocyanins. Wild genotypes generally contain more antioxidants than cultivated counterparts. So, seven different antioxidants assays on extracts from cultivated and wild Vaccinium berries were performed, to evaluate their difference in terms of bioactivity on oxidative protection and minimum dosage to have a significant action. MATERIALS AND METHODS: Four cell-free antioxidant assays (ABTS radical scavenging and electronic paramagnetic resonance using Fremy's salt, superoxide anion and hydroxyl radical), and three assays on human cells (two luminol amplified chemiluminescence, LACL, one on DNA damage, COMET) were used to measure the effects of cultivated blueberry (V. corymbosum) and wild bilberry (V. myrtillus) on the differently induced oxidative stress. Concentrations vs activity patterns were obtained by successive dilutions of extracts in order to identify both EC50 and minimum significant activity (MSA). RESULTS: All the assays (except for the hydroxyl radical scavenging) showed a good relationship mainly with anthocyanin and polyphenol content and the significant greater activity of wild Vaccinium extracts. In fact, LACL data gave an EC50 of 11.8 and an MSA of 5.2 g were calculated as fresh weight dosage in cultivated berries, compared with lower doses in wild berries, EC50 of 5.7 g and MSA of 3.4 g. CONCLUSIONS: Wild Vaccinium extracts averaged 3.04 and 2.40 fold more activity than cultivated extracts by EC50 and MSA, respectively. COMET assay confirmed the stronger action on DNA protection in wild samples.

The deduced structure of the T cell receptor gamma locus in Canis lupus familiaris.

Analyzing the recent high-quality genome sequence of the domestic dog (Canis lupus familiaris), we deduced for the first time in a mammalian species belonging to Carnivora order, the genomic structure and the putative origin of the TRG locus. New variable (TRGV), joining (TRGJ) and constant (TRGC) genes for a total of 40 are organized into eight cassettes aligned in tandem in the same transcriptional orientation, each containing the basic recombinational unit V-J-J-C, except for a J-J-C cassette, that lacks the V gene and occupies the 3' end of the locus. Amphiphysin (AMPH) and related to steroidogenic acute regulatory protein D3-N-terminal like (STARD3NL) genes flank, respectively, the 5' and 3' ends of the canine TRG locus that spans about 460kb. Moreover LINE1 elements, evenly distributed along the entire sequence, significantly (20.59%) contribute to the architecture of the dog TRG locus. Eight of the 16 TRGV genes are functional and belong to 4 different subgroups. Canine TRGJ genes are two for each cassette and only seven out of 16 are functional. The germline configuration and the exon-intron organization of the 8 TRGC genes was determined, six of them resulting functional. The dot plot similarity genomic comparison of human, mouse and dog TRG loci highlighted the occurrence of reiterated duplications of the cassettes during the dog TRG locus evolution. On the other hand the low ratio of functional genes to the total number of canine TRG genes (21/40), suggest that there is no correlation between the extensive duplications of the cassettes and a need for new functional genes. Furthermore the comparison revealed that the TRGC6, C7 and C8 genes are highly related across species suggesting these existed before the primate-rodent-canidae lineages diverged.

Organization, structure and evolution of 41kb of genomic DNA spanning the D-J-C region of the sheep TRB locus.

A genomic region of 41,045 bp encompassing the 3'-end of the sheep T cell receptor beta chain was sequenced. Extensive molecular analysis has revealed that this region retains a unique structural feature for the presence of a third D-J-C cluster, never detected in any other mammalian species examined so far. A total of 3 TRBD, 18 TRBJ and 3 substantially identical TRBC genes were identified in about 28kb. At 13kb, downstream from the last TRBC gene, in an inverted transcriptional orientation, lies a TRBV gene. Sequence comparison and phylogenetic analyses have demonstrated that the extra D-J-C cluster originated from an unequal crossing over between the two ancestral TRBC genes. Interspersed repeats spanning 22.2% of the sequence, contribute to the wider size of the sheep TRB locus with respect to the other mammalian counterparts, without modifying the general genomic architecture. The nucleotide and predicted amino acid sequences from peripheral T cells cDNA clones indicated that the genes from cluster 3 are fully implicated in the beta chain recombination machinery. Closer inspections of the transcripts have also shown that inter-cluster rearrangements and splice variants, involving the additional cluster, increase the functional diversity of the sheep beta chain repertoire.

Decline in verbal memory in non-demented older adults.

Decline in memory function was detected in 30% of healthy community-dwelling elderly over 6 years using a task assessing delayed word list recall. Individuals with memory decline over time also demonstrated relative deficits on additional tasks of memory and learning, a task of working memory and executive function, and on a verbal (category) fluency task at their most recent assessment. These relative deficits in the performance of individuals with memory decline cannot be explained by age-related changes, education, intelligence, mood, health-related factors, or the individuals' APOE epsilon 4 status. Decline in memory performance did not result in greater complaints of cognitive difficulties when compared with normal elderly, nor did it limit overall participation in life activities. Although the significance of memory decline in the current study was not determined quantitatively, memory decline is consistent with the early deterioration characteristic of mild cognitive impairment and preclinical Alzheimer's disease and confirms the need to monitor individuals with objective memory decline, even when these individuals fall within normal limits for a given neuropsychological task.

Artiodactyl emergence is accompanied by the birth of an extensive pool of diverse germline TRDV1 genes.

Molecular cloning of cDNA from gamma/delta T cells has shown that in sheep, the variable domain of the delta chain is chiefly determined by the expression of the TRDV1 subgroup, apparently composed of a large number of genes. There are three other TRDV subgroups, but these include only one gene each. To evaluate the extent and the complexity of the genomic TRDV repertoire, we screened a sheep liver genomic library from a single individual of the Altamurana breed and sheep fibroblast genomic DNA from a single individual of the Gentile di Puglia breed. We identified a total of 22 TRDV1 genes and the TRDV4 gene. A sequence comparison between germline and the rearranged genes indicates that, in sheep, the TRDV repertoire is generated by the VDJ rearrangement of at least 40 distinct TRDV1 genes. All germline TRDV1 genes present a high degree of similarity in their coding as well as in 5' and 3' flanking regions. However, a systematic analysis of the translation products reveals that these genes present a broadly different and specific repertoire in the complementarity-determining regions or recognition loops, allowing us to organize the TRDV genes into sets. We assume that selection processes operating at the level of ligand recognition have shaped the sheep TRDV germline repertoire. A phylogenetic study based on a sequence analysis of the TRDV genes from different mammalian species shows that the diversification level of these genes is higher in artiodactyl species compared to humans and mice.

Evolution of TRG clusters in cattle and sheep genomes as drawn from the structural analysis of the ovine TRG2@ locus.

The availability of genomic clones representative of the T-cell receptor constant gamma (TRGC) ovine genes enabled us to demonstrate, by fluorescent in situ hybridization (FISH) on cattle and sheep metaphases, the presence of two T-cell receptor gamma (TRG1@ and TRG2@) paralogous loci separated by at least five chromosomal bands on chromosome 4. Only TRG1@ is included within a region of homology with human TRG locus on chromosome 7, thus TRG2@ locus appears to be peculiar to ruminants. The structure of the entire TRG2@ locus, the first complete physical map of any ruminant animal TCR gamma locus, is reported here. The TRG2@ spans about 90 kb and consists of three clusters that we named TRG6, TRG2, and TRG4, according to the constant genes name. Phylogenetic analysis has highlighted the correlation between the grouping pattern of cattle and sheep variable gamma (TRGV) genes and the relevant TRGC; variable (V), joining (J), and constant (C) rearrange to be found together in mature transcripts. The simultaneous results on the TRG2@ locus molecular organization in sheep and on the phylogenetic analysis of cattle and sheep V expressed sequences indicate that at least six TRG clusters distributed in the two loci are present in these ruminant animals. The inferred evolution of TRG clusters in cattle and sheep genomes is consistent with a scenario where a minimal ancient cluster, containing the basic structural scheme of one V, one J, and one C gene, has undergone a process of duplication and intrachromosomal transposition.

Memory decline in healthy older people: implications for identifying mild cognitive impairment.

BACKGROUND: Criteria for mild cognitive impairment require objective evidence of a memory deficit but do not require objective evidence of memory decline. Application of these criteria may therefore result in the misclassification of older patients with memory decline as normal because their neuropsychological test performance at a single point in time may be within normal limits. This study aimed to identify and characterize older people with memory decline. METHOD: Word list delayed recall (WLDR) test performance was assessed on five occasions during a 2-year period in a cohort of healthy older individuals. Older people with declining (n = 35) and nondeclining (n = 66) WLDR scores were identified. Both subgroups were then compared on apoE genotype, Clinical Dementia Rating, and neuropsychological test performance at the fifth assessment. RESULTS: Thirty-four percent of the group with declining memory recorded a Clinical Dementia Rating of 0.5, compared with 5% of the nondeclining memory group. No between-group differences were observed in cognitive domains other than memory, self-reported cognitive failures, or the proportion of each group carrying the apoE epsilon 4 allele. CONCLUSIONS: A large proportion of healthy older individuals show memory decline, which may represent the early stages of a potentially more severe cognitive impairment. Further investigation is necessary to determine the relationship between apoE genotype, self-reported cognitive impairment, and memory decline in older people.

Genomic organization of sheep TRDJ segments and their expression in the delta-chain repertoire in thymus.

cDNA sequences obtained from polymerase chain reaction products of reverse-transcribed RNA from sheep thymus showed the presence of a large number of members of the TRDV1 gene family. Some are TRDV1 genes also found in peripheral blood lymphocytes, while four genes had not been described so far. The cDNA sequences also showed extensive junctional diversity and a preferential usage of the three TRDJ elements. We characterized the genomic organization of the sheep TRDJ locus and detected a correlation between the nonrandom usage of TRDJ elements during development and their chromosomal order.

Norms and the effects of demographic variables on a neuropsychological battery for use in healthy ageing Australian populations.

OBJECTIVE: The current study examined the performance of a healthy ageing population on the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neuropsychological test battery in order to determine norms for use in an Australian setting. The effects of age, education, gender and mood on cognitive performance in healthy older individuals were also explored. METHOD: The CERAD neuropsychological battery was administered to a sample of healthy elderly subjects (n = 243). Subjects also completed an anxiety inventory and a depression scale. Means and standard deviations of different age, gender and education groups are reported as normative data. A Principal Components Analysis (PCA) was also calculated. Linear regression was applied to the five factors extracted from the PCA using age, education, gender and mood as independent variables. RESULTS: All recorded means were within 1 SD of those reported in the original CERAD normative study. Five factors that loaded on measures of memory and learning, language, praxis and executive function were extracted. The independent variables age, education and gender all had significant effects on cognitive performance. However, mood had no such effect. CONCLUSIONS: Risk factors for cognitive decline indicated by the CERAD battery include age, education and gender. Anxiety and depression are not associated with CERAD cognitive performance. The CERAD battery is a valid and reliable neuropsychological tool that may assist in the detection and diagnosis of Alzheimer's disease in Australian populations.

The effects of age and mood on saccadic function in older individuals.

To investigate the effect of age and mood on saccadic function, we recorded prosaccades, predictive saccades, and antisaccades from 238 cognitively normal, physically healthy volunteers aged 44 to 85 years old. Mood levels were measured using the State-Trait Anxiety Inventory and Center for Epidemiological Studies Depression Scale inventories. Small, but significant, positive relationships with age were observed for the mean latency and associated variability of latency for all types of saccades, as well as the antisaccade error rate. Saccade velocity or accuracy was unaffected by age. Increasing levels of depression had a minor negative influence on the antisaccade latency, whereas increasing levels of anxiety raised the antisaccade error rate marginally.

Collection and normal levels of the amyloid precursor protein in plasma.

The amyloid precursor protein is contained in platelet alpha granules and released with degranulation. Methods are described to control for amyloid precursor protein release from platelets during blood collection and processing. In normal subjects (n = 97; age range, 44-84 years), the average plasma level of amyloid precursor protein was 6.5 +/- 1.8 ng/ml.

Assignment of the TCRA/TCRD locus to sheep chromosome bands 7q1.4-->q2.2 by fluorescence in situ hybridization.

Two genomic clones for the TCRA/TCRD locus have been isolated and characterized in sheep. The first clone corresponds to a new sheep Valpha element, the other to the entire Cdelta gene. Chromosomal mapping by fluorescence in situ hybridization (FISH) of Valpha and Cdelta clones localized the TCRA/TCRD locus on sheep chromosome region 7q14-->q22. This is the first physical assignment for genomic clones on sheep chromosome 7 by FISH. Moreover, the present data put the ovine 7q14-->q22 and the human 14q11.2 regions in the same syntenic group.

Structural organization of multiple alphoid subsets coexisting on human chromosomes 1, 4, 5, 7, 9, 15, 18, and 19.

FISH experiments on metaphase chromosomes, interphase nuclei, and extended chromatin were performed to investigate the structural organization of alphoid subsets coexisting on human chromosomes 1, 4, 5, 7, 9, 15, 18, and 19. Results indicate that multiple subsets present on chromosomes 5, 7, 15, 18, and 19 are organized in structurally distinct and contiguous domains, while those on chromosomes 4 and 9 give perfectly overlapping signals. Chromosome 1 shows a peculiar organization: probe pAL1, specific for this chromosome, detects two distinct domains separated by the subset identified by probe pZ5.1. The order along the chromosome of alphoid subsets lying on chromosomes 5, 7, 15, 18, and 19, organized in distinct blocks, has also been established. The relationship between the structural organization of these alphoid sequences and their evolutionary history in great apes is discussed.

Combined paresis and restriction of the extraocular muscles after orbital fracture: a study of 16 patients.

Sixteen patients with combined paresis and restriction of extraocular muscle(s) orbital fracture repair were studied before and after in order to determine the clinical features and management of such patients. All 16 patients showed limited ductions of the involved eye in the field of action of the entrapped, paretic muscle and of the antagonist muscle after orbital fracture. Single extraocular muscles (13 patients) and two extraocular muscles (three patients) were demonstrated adjacent to the fracture site on orbital computed tomography (CT). In three patients prior to orbital surgery, a deviation in primary position was present. After fracture repair with release of the entrapped muscle in all patients, evidence of paresis of the muscle was demonstrated by underaction in its field of action and overaction in the field of its antagonist. There was a resultant manifest tropia or phoria in the primary position. In seven patients, the paresis gradually improved with no tropia and little diplopia in the functional fields of gaze. Three patients had minimal deviations and required no further treatment. Six patients with significant deviations required prisms (three patients) or strabismus surgery (three patients). The latter three patients had two muscles involved. Results of this study demonstrate that the ophthalmologist must appropriately diagnose patients with paresis and restriction of an extraocular muscle and counsel them that "new" diplopia may occur after orbital fracture repair and that this diplopia may require additional therapy.

Duplication of a gene-rich cluster between 16p11.1 and Xq28: a novel pericentromeric-directed mechanism for paralogous genome evolution.

We have identified a 26.5 kb gene-rich duplication shared by human Xq28 and 16p11.1. Complete comparative sequence analysis of cosmids from both loci has revealed identical Xq28 and 16p11.1 genomic structures for both the human creatine transporter gene (SLC6A8) and five exons of the CDM gene (DXS1357E). Overall nucleotide similarity within the duplication was found to be 94.6%, suggesting that this interchromosomal duplication occurred within recent evolutionary time (7-10 mya). Based on comparisons between genomic and cDNA sequence, both the Xq28 creatine transporter and DXS1357E genes are transcriptionally active. Predicted translation of exons and RT-PCR analysis reveal that chromosome 16 paralogs likely represent pseudogenes. Comparative fluorescent in situ hybridization (FISH) analyses of chromosomes from various primates indicate that this gene-rich segment has undergone several duplications. In gorilla and chimpanzee, multiple pericentromeric localizations on a variety of chromosomes were found using probes from the duplicated region. In other species, such as the orangutan and gibbon, FISH signals were only identified at the distal end of the X chromosome, suggesting that the Xq28 locus represents the ancestral copy. Sequencing of the 16p 11.1/Xq28 duplication breakpoints has revealed the presence of repetitive immunoglobulin-like CAGGG pentamer sequences at or near the paralogy boundaries. The mobilization and dispersal of this gene-rich 27 kb element to the pericentromeric regions of primate chromosomes defines an unprecedented form of recent genome evolution and a novel mechanism for the generation of genetic diversity among closely related species.

A transcription map in the CATCH22 critical region: identification, mapping, and ordering of four novel transcripts expressed in heart.

The acronym CATCH22 is used to indicate collectively a group of related phenotypes, namely velocardiofacial syndrome (VCFS), DiGeorge anomaly (DGA), and conotruncal anomaly face, which are associated with deletions within 22q11.2 in the great majority of patients. A deletion map has allowed us to delimit a smallest region of deletion overlap, considerably smaller than the commonly deleted region. We have mapped within this region the chromosomal breakpoint of a balanced translocation patient presenting with a DGA/VCFS phenotype, making this region the strongest candidate for the location of the gene(s) responsible for the disease phenotype. We report a systematic gene search in this region and show the presence of at least six distinct transcripts, two of which have been previously described. The region searched was approximately 270 kb; therefore, an average of one transcript every 45 kb was found. We generated eight new ESTs and mapped two ESTs present in public databases. All six transcripts are expressed in heart, an organ involved in 70%-80% of CATCH22 patients. We show that the multimethod approach to search for expressed sequences is effective and indeed necessary for a comprehensive search and provides molecular tools for further characterization of the potential genes identified.