Deletion of the Cochliobolus heterostrophus mating-type (MAT ) locus promotes the function of MAT transgenes

Cochhobolus heterostrophus has alternate genes (MAT-1 andMAT-2) at its mating-type locus. Transformants of aMAT-1 or aMAT-2 strain carrying a transgene of opposite mating type can self and are dual maters; the transgene, however, promotes development of pseudothecia only, not ascospores. To determine if the resident gene interferes with the function of the transgene, transformation vectors were designed to delete different amounts (2.5 kb, 5.7 kb, and 6.3 kb) of DNA at theMAT locus. Deletions occurred at a higher frequency (about 90% of transformants) with linearized plasmid than with circular plasmid (about 15% of transformants), and all three vectors were equally efficient at gene replacement. BothMAT-1 andMAT-2 could be deleted with the same set of vectors. Re-transformation of deletion strains (regardless of deletion size) with a wild-type copy ofMAT restored full mating ability, indicating that the residentMAT gene interferes with function of theMAT transgene. Moreover, sexual development was normal whether theMAT transgene integrated at the homologous or at an ectopic site.     

Haplotype structure,LD blocks,and uneven recombination within the LRP5 gene

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.     

Prostate implant evaluation using tumour control probability--the effect of input parameters

In this paper, we examine the effect of treatment parameters in a model used to evaluate permanent prostate implants. The model considers the prostate to be composed of 12 sub-sections, each sub-section is assigned a cell density based on the probability of finding cancer foci in that sub-section. Wasted dose as a result of the dose rate from the implant falling below a level adequate to counteract repopulation was found to vary by 2-16% over the range of radiosensitivity and repopulation rates considered. Within the model, applied to five dose distributions, the uncertainty in the tumour control probability (TCP) values calculated for each sub-section as a result of differences in the model parameters, was found to be less than 12% in most cases for the good quality implants. The difference in TCP values was much larger for the poor quality implant. Substituting a heterogeneous distribution of alpha for a single mean value resulted in generally lower TCP values though introducing a cutoff value with a Gaussian distribution had a profound effect on the calculated values. Despite uncertainties in the parameters, the model was able to identify sub-sections at risk of local recurrence but as a result of these uncertainties, the TCP values can only be considered in the relative rather than absolute sense.     

Functional characteristics of coronary vasomotor function following intramyocardial gene therapy with naked DNA encoding for vascular endothelial growth factor165.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. VEGF gene therapy improves perfusion of ischemic myocardium in experimental models and possibly in patients with end-stage coronary artery disease. In addition to its proliferative and migratory effect on endothelial cells, it also activates and up-regulates endothelial nitric oxide synthase (eNOS). Therefore, the authors investigated coronary endothelium-dependent vasodilatation in patients before and after VEGF gene therapy. The effect of intracoronary acetylcholine infusion on coronary diameter was assessed at baseline and after 3 months follow-up in patients with end-stage coronary artery disease treated with VEGF gene and in controls scheduled for elective percutaneous transluminal coronary angioplasty (PTCA) (acetylcholine test at diagnostic angiography and before a subsequently scheduled PTCA). Five out of six VEGF patients experienced a reduction in anginal complaints. Angiographic evidence for improved collateral filling was evident in two out of six patients. The vasoconstrictive response to acetylcholine was partly converted into dilatation. In contrast, the acetylcholine response in control patients remained vasoconstrictive. In conclusion, VEGF gene therapy has an important beneficial effect on the functional characteristics of the myocardial vascular network. Therefore, this therapy can potentially play an important role in all stages of the atherosclerotic process.     

The Secreted Larval Acidic Proteins (SLAPs) of Onchocerca spp. are encoded by orthologues of the alt gene family of Brugia malayi and have host protective potential

Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and the causative agent of onchocerciasis, or 'River Blindness'. Resistance to infection is associated with immune responses to the infective, third-stage (L3) larvae. The antigens of greatest interest for their vaccine potential are surface and secreted molecules. We have previously identified a family of Secreted Larval Acidic Proteins (SLAPs) from the L3 larvae of O. volvulus by biosynthetic labelling. Here, we provide further characterisation of these molecules following cloning and expression of the corresponding cDNAs. Using protein sequencing, we show that SLAPs are members of the alt gene family, first described in the lymphatic filarial parasite, Brugia malayi. Ov-ALT-1 and Ov-ALT-2 correspond with 20 and 18kDa SLAPs. Both proteins are highly acidic and related by sequence, differing chiefly in an 8-amino acid deletion from Ov-ALT-2. By immunochemistry, we confirm that Ov-ALTs are highly stage-specific, being expressed exclusively in late L2 and L3 larvae during growth in the vector. They are synthesised and stored in the glandular oesophagus. Secretion is triggered by the resumption of development in the definitive host and occurs via the pseudocoelom and cuticle. Serological responses in humans to recombinant Ov-ALT-1 indicate that the level of IgG production may be governed by the force of transmission but does not overtly reflect infection status. Immunisation of mice with recombinant Ov-ALT-1 resulted in a modest level of protection against challenge with O. volvulus L3 larvae (P = 0.036). We conclude that Ov-ALT genes, like those of other filariae, are of interest from the standpoint of parasite transmission and infectivity. They may also offer promise as components of a future sub-unit vaccine should the means to enhance protection be achieved.     

The matrix metalloproteinase-3 (MMP-3) 5A/6A promoter polymorphism is not associated with ischaemic heart disease: analysis employing a family based approach

Matrix metalloproteinase-3 (MMP-3) has been proposed as an important mediator of the atherosclerotic process. The possible role of the functional -1612 5A/6A polymorphism of the MMP-3 gene in the susceptibility to ischaemic heart disease (IHD) was investigated in a well-defined Irish population using two recently described family based tests of association. One thousand and twelve individuals from 386 families with at least one member prematurely affected with IHD were genotyped. Using the combined transmission disequilibrium test (TDT)/sib-TDT and the pedigree disequilibrium test (PDT), no association between the MMP-3 -1612 5A/6A polymorphism and IHD was found. Our data demonstrate that, in an Irish population, the MMP-3 -1612 5A/6A polymorphism is not associated with IHD.     

Modification of T-cell receptor Vβ repertoire in response to allergen stimulation in peanut allergy

Background: Peanut is one of the most common foods causing allergic reactions and is the most common cause of fatal and near-fatal food-related anaphylaxis. Little is known of the immunologic mechanisms that underlie peanut allergy. Objectives: In this study we examined clonality of the T-cell response (TCR) to peanut in MHC class II identical, peanut allergy–discordant sibling pairs. Methods: Four sibling pairs were investigated. The TCR repertoire was analyzed before and after in vitro stimulation of PBMCs with crude peanut or PHA, as control for general/nonspecific reactivity. Eighteen TCR-Vβ families were examined by flow cytometry. Where significant differences in incidence of particular TCR-Vβ families were observed, PCR familyspecific cDNA amplification and gene scanning were performed. Results: After stimulation with peanut, no selective expansion of any TCR-Vβ subpopulation was observed with flow cytometry, in either the peanut-allergic or nonallergic siblings, with the exception of 1 peanut-allergic subject who demonstrated a significant increase of TCR-Vβ11⁺ cells (0.3%-5.9% of the total CD3⁺ cells). However, gene scanning revealed predominant single-size PCR products for TCRBV11 in all peanut-allergic subjects after peanut stimulation. TCRBV11 polyclo-nality was observed in allergic and nonallergic subjects before peanut stimulation and in nonallergic subjects after peanut stimulation. In comparison, all subjects, before and after stimulation with peanut, showed polyclonality for TCRBV2.Conclusions: Our results argue for clonal or oligoclonal TCRs to crude peanut and indicate that changes in the TCRBV11 subpopulation are restricted to peanut-allergic subjects after stimulation with crude peanut allergen. (J Allergy Clin Immunol 2001;107:1089-94.)     

Binding of influenza viruses to sialic acids: reassortant viruses with A/NWS/33 hemagglutinin bind to alpha2,8-linked sialic acid

We have examined the specificity of binding of A/NWS/33 hemagglutinin (HA), exploring the effects of fucosylation, changing the Gal-GlcNAc linkage between the second and third sugars, and binding affinity for alpha2,8-linked sialic acid. The HA of A/NWS/33(HA)-Tokyo/67(NA) (NWS-Tok, H1N2) virus binds to 3'-linked sialyllactose with 10-fold higher affinity than 3' sialyllactosamine and 3-fold higher affinity than 6' sialyllactosamine. The P227H mutation in A/NWS/33(P227H)(HA)-A/Memphis/31/98(NA) (NWS-Mem/98, H1N2) results in sevenfold lower affinity for 3' sialyllactose, but binding to 6' sialyllactosamine is unchanged. The apparent switch from 3' to 6' specificity is solely due to a loss of Siaalpha2,3 binding. Fucosylation of the third sugar and changing the linkage between second and third sugars had little effect on binding by NWS-Tok, but marked effects on A/NWS/33(P227H)(HA)-tern/Australia/G70c/75(NA) (NWS-G70c, H1N9) and NWS-Mem/98. NWS-Tok, NWS-G70c, and NWS-Mem/98 bind to alpha2,8-bisialic acid with high affinity. NWS-Mem/98 can also bind to alpha2,8-trisialic acid, but with lower affinity. Together, these data show that alpha2,8-linked sialic acid, fucosylation of the third sugar, and linkage between the second and third sugars could play important roles in allowing efficient virus binding to its host cell. The finding that influenza viruses have the potential to bind to alpha2,8-linked sialic acid is a new influenza virus-receptor interaction pathway.