Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3

Genetic linkage studies have indicated that chromosome 14q24.3harbours a major locus for early-onset (onset age <65 years)Alzheimer's disease (AD3). Positional cloning efforts have identifieda novel gene S182 or presenilin 1 as the AD3 gene. We have mappedS182 in the AD3 candidate region between D14S277 and D14S284defined by genetic linkage studies in the two chromosome 14linked, early-onset AD families AD/A and AD/B. We have shownthat S182 is expressed in lymphoblasts and have determined thecomplete cDNA in both brain and lymphoblasts by RT-PCR sequencing.S182 is alternatively spliced in both brain and lymphoblastswithin a putative phosphorylation site located 5' in the codingregion. We identified two novel mutations, Ile143Thr and Gly384Alalocated in, respectively, the second transmembrane domain andin the sixth hydrophilic loop of the putative transmembranestructure of S182. As families AD/A and AD/B have a very similarAD phenotype our observation of two mutations in functionallydifferent domains suggest that onset age and severity of ADmay not be very helpful predictors of the location of putativeS182 mutations.     

Microarray-based comparative genomic analyses of the human malaria parasite Plasmodium falciparum using Affymetrix arrays

Microarray-based comparative genomic hybridization (CGH) provides a powerful tool for whole genome analyses and the rapid detection of genomic variation that underlies virulence and disease. In the field of Plasmodium research, many of the parasite genomes that one might wish to study in a high throughput manner are not laboratory clones, but clinical isolates. One of the key limitations to the use of clinical samples in CGH, however, is the miniscule amounts of genomic DNA available. Here we describe the successful application of multiple displacement amplification (MDA), a non-PCR-based amplification method that exhibits clear advantages over all other currently available methods. Using MDA, CGH was performed on a panel of NF54 and IT/FCR3 clones, identifying previously published deletions on chromosomes 2 and 9 as well as polymorphism in genes associated with disease pathology.     

Expression of transient receptor potential C6 and related transient receptor potential family members in human airway smooth muscle and lung tissue

Elevation of the intracellular free Ca(2+) concentration regulates many functional responses in airway smooth muscle, including contraction, proliferation, adhesion, and cell survival. This increase in calcium can be achieved by a release from internal stores (sarcoplasmic reticulum) and/or entry across the cell membrane from the extracellular environment. The molecular identity of this calcium influx pathway in human airway smooth muscle (HASM) remains unclear. Functional studies using Fluo 4-loaded HASM suggest the presence of a histamine H(1) receptor-activated Ca(2+) entry pathway with characteristics similar to those seen with transient receptor potential (TRP) family homologs. Using a range of molecular and cell biological approaches we defined the expression pattern of transient receptor potential classics (TRPC) homologs in airway cells and tissue. Here we show that HASM and human bronchial epithelial cells both express TRPC1, -4, and -6, with HASM also expressing TRPC3 at the mRNA level. Identification of TRPC6 protein by western blot and confocal microscopy indicated that the protein is localized in specific cell types, suggesting that it plays an important role in regulating key functions in airway cells. These data demonstrate the expression of a range of TRPC homologs in the airway and the presence of a functional Ca(2+) entry pathway with characteristics typical of TRPC family members. TRPC homologs may provide an important novel target for the treatment of airway disease.     

Diversity of enteric viruses detected in patients with gastroenteritis in a tertiary referral paediatric hospital

The genetic diversity of enteric viruses co-circulating in a cohort of patients with viral gastroenteritis in a large tertiary paediatric hospital in London, UK, was determined. Multiple strains of noroviruses (NV), sapoviruses (SV) and astroviruses (HAsV) were detected in these patients, indicating the likelihood of multiple introductions from different sources, possible sub-clinical infections and simultaneous infection with different viruses in immunocompromised and other patients. Routine screening of immunocompromised patients and infection control procedures are important to prevent nosocomial infection.     

Failure to produce conditioning with low-dose trimethylthiazoline or cat feces as unconditioned stimuli

Trimethylthiazoline (TMT), a derivative of fox feces, has been reported to fail to produce aversive conditioning as an unconditioned stimulus (UCS) when presented in large amounts (I. S. McGregor, L. Schrama, P. Ambermoon, & R. A. Dielenberg, 2002). Experiment I evaluated very low TMT levels that nonetheless produced defensive behaviors in rats during exposure. Although each level (0.01, 0.05, and 0.10 microl TMT) produced significant change in defensiveness, none resulted in significant changes the following day in the absence of TMT. Experiment 2 evaluated cat urine, cat feces, and cat fur/skin odor against a no-odor control. Urine produced no significant changes, but feces and fur/skin odors elicited virtually identical changes in defensive behaviors during exposure. When tested the next day in the absence of odor, the fur/skin odor-exposed group showed significant differences on the same behaviors as during exposure, but the feces-exposed group showed no differences on any measure. Results suggest that lack of conditioning to TMT may relate to the type of predator odor rather than the amount, predator species, or possible lack of odor components in TMT that are present in natural feces. Predator feces may also be less effective as a UCS because they are poorly predictive of the actual presence of the predator, suggesting the need for a reevaluation of UCS functions in aversive conditioning.     

Genetic heterogeneity of small ruminant lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-strain-based immunoassay

The pol and gag gene fragments of small ruminant lentivirus field isolates collected in the last decade in Italy were amplified, sequenced, and analyzed. Phylogenetic analysis revealed that the majority of ovine isolates form a distinct cluster more similar to caprine lentivirus prototypes than to the visna virus prototype. These findings confirm and extend those reported by Leroux et al. (Arch. Virol., 142:1125-1137, 1997). Moreover, we observed that a variable region of Gag, included in the fragment analyzed, corresponded to one of the three major capsid antigen epitopes, which suggests that the antibody response to this epitope may be type specific. To test this hypothesis, two recombinant peptides, derived from the Icelandic prototype K1514 and this novel genotype, were expressed and used in an enzyme-linked immunosorbent assay to screen a panel of ovine and caprine sera collected from different geographical locations in Italy. Several sera reacted in a type-specific manner, indicating that in a diagnostic setting the combination of at least these two type-specific peptides is necessary to cover a wide range of infections. Additionally, these results support the hypothesis of cross-species transmission based on the phylogenetic analysis described above. This has implications for the control and eradication of small ruminant lentivirus infections.     

Thyroid hormone promotes neurogenesis in the Xenopus spinal cord.

Three phases of neurogenesis can be recognized during Xenopus spinal cord development. An early peak during gastrulation/neurulation is followed by a phase of low level neurogenesis throughout the remaining embryonic stages and a later peak at early larval stages. We show here that several genes known to be essential for early neurogenesis (X-NGNR-1, XNeuroD, XMyT1, X-Delta-1) are also expressed during later phases of neurogenesis in the spinal cord, suggesting that they are involved in regulating spinal neurogenesis at later stages. However, additional neuronal determination genes may be important during larval stages, because X-NGNR-1 shows only scant expression in the spinal cord during larval stages. Thyroid hormone treatment of early larvae promotes neurogenesis in the spinal cord, where thyroid hormone receptor xTRalpha is expressed from early larval stages onward and results in precocious up-regulation of XNeuroD, XMyT1, and N-Tubulin expression. Similarly, thyroid hormone treatments of Xenopus embryos, which were coinjected with xTRalpha and the retinoid X receptor xRXRalpha, repeatedly resulted in increased numbers of neurons, whereas unliganded receptors repressed neurogenesis. Our findings show that thyroid hormones are sufficient to up-regulate neurogenesis in the Xenopus spinal cord.     

The Bioperl toolkit: Perl modules for the life sciences

The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort.