Isolation of DNA from the centromere of human chromosome 7 by microdissection

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a micro-dissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3–4 cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.This revised version was published online in November 2005 with corrections to the Cover Date.     

Mitochondrial DNA in the aquatic fungus Allomyces

Summary The mitochondrial DNA from seven species of the aquatic phycomycete Allomyces has been isolated and characterized by restriction enzyme analysis. Comparison of the mitochondrial DNA restriction enzyme fragmentation patterns showed pronounced differences not only among species but also among four isolates of A. arbuscula. The mitochondrial DNAs range in size from 39 kbp in A. neo-moniliformis to 56 kbp in A. macrogynus.A physical map of the mitochondrial DNA of Allomyces arbuscula strain Costa Rica 21 has been constructed. The genome is circular and has a size of 49.2 kbp. The genes coding for the small and large ribosomal RNAs, cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, and ATPase subunits 6 and 9 were localized in the mitochondria) DNA by heterologous hybridization with specific mitochondria) gene probes from Saccaromyces cerevisiae and Neurospora crassa. Comparison of the gene map of the closely related species Blastocladiella emersonii with that of A. arbuscula indicates a similar gene order in the two organisms.     

Rapid PCR Detection of Helicobacter pylori-Associated Virulence and Resistance Genes Directly from Gastric Biopsy Material

We have developed a PCR-based method to detect macrolide resistance and the virulence gene cagA in Helicobacter pylori within 24 h, thereby improving the lengthy process of culture-based approaches. Total DNA was prepared directly from stomach biopsy specimens. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.     

Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis

Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost "on top" of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.     

Giant ragweed specific immunotherapy is not effective in a proportion of patients sensitized to short ragweed: analysis of the allergenic differences between short and giant ragweed

BACKGROUND: Short ragweed and giant ragweed pollen allergens are considered largely cross-reactive, and it is generally believed that 1 species is sufficient for skin testing and immunotherapy. However, in the area north of Milan (a zone widely invaded only by short ragweed), about 50% of patients submitted to injection specific immunotherapy with giant ragweed showed little or no clinical response, but showed an excellent outcome if they were shifted to short ragweed specific immunotherapy. OBJECTIVE: To investigate allergenic differences between short and giant ragweed. METHODS: IgE reactivity to short ragweed of sera from 16 patients allergic to ragweed was assessed by immunoblot before and after absorption with short and giant ragweed. Moreover, 41 ragweed-monosensitive patients underwent skin prick test with both ragweed species. RESULTS: In several cases, preabsorption of sera with giant ragweed extract was unable to inhibit IgE reactivity fully against both a 43-kd allergen and other allergens at different molecular weights in short ragweed. On skin prick test, short ragweed induced larger wheals than giant ragweed in the majority of patients, and 6 of 41 (15%) patients were strongly short ragweed-positive but giant ragweed-negative. The immunoblot with the serum from 1 of these subjects showed a strong IgE reactivity to short ragweed at about 43 kd in the absence of any reactivity to giant ragweed. CONCLUSION: Short and giant ragweed are not allergenically equivalent. Allergenic differences involve both the major allergens Amb a 1-2/Amb t 1-2 and some minor allergens. In patients allergic to ragweed, both diagnosis in vivo and immunotherapy should always be performed by using the ragweed species present in that specific geographic area.     

Prediction of the average skin temperature in warm and hot environments

The prediction of the mean skin temperature used for the Required Sweat Rate index was criticised for not being valid in conditions with high radiation and high humidity. Based on a large database provided by 9 institutes, 1999 data points obtained using steady-state conditions, from 1399 experiments and involving 377 male subjects, were used for the development of a new prediction model. The observed mean skin temperatures ranged from 30.7 °C to 38.6 °C. Experimental conditions included air temperatures (T _a) between 20 and 55 °C, mean radiant temperatures (T _r) up to 145 °C, partial vapour pressures (P _a) from 0.2 to 5.3 kPa, air velocities (v _a) between 0.1 and 2 m/s, and metabolic rates (M) from 102 to 620 W. Rectal temperature (T _re) was included in the models to increase the accuracy of prediction. Separate models were derived for nude (clothing insulation, I_cl, ≤0.2 clo, where 1 clo=0.155 m² · °C · W⁻¹, which is equivalent to the thermal insulation of clothing necessary to maintain a resting subject in comfort in a normally ventilated room, air movement=10 cm/s, at a temperature of 21 °C and a humidity of less than 50%) and clothed (0.6 ≤ I_cl ≤ 1.0 clo) subjects using a multiple linear regression technique with re-sampling (non-parametric bootstrap). The following expressions were obtained for nude and clothed subjects, respectively: T _sk=7.19 + 0.064T _a + 0.061T _r + 0.198P _a− 0.348v _a + 0.616T _re and T _sk=12.17 + 0.020T _a + 0.044T _r + 0.194P _a − 0.253v _a + 0.0029M + 0.513T _re. For the nude and clothed subjects, 83.3% and 81.8%, respectively, of the predicted skin temperatures were within the range of ±1 °C of the observed skin temperatures. It is concluded that the proposed models for the prediction of the mean skin temperature are valid for a wide range of warm and hot ambient conditions in steady-state conditions, including those of high radiation and high humidity. Accepted: 7 February 2000     

Activation induces apoptosis in Herpesvirus saimiri-transformed T cells independent of CD95 (Fas,APO-1)

Signaling via the T cell receptor (TCR)/CD3 complex of pre-activated T cells induces apoptosis. Such an activation-induced cell death (AICD) is thought to play an important role in the regulation of cellular immune responses. In this study we analyzed pathways of AICD by using human T cells transformed by Herpesvirus saimiri. These growth-transformed T cells show the phenotype of activated mature T cells and continue to express a functionally intact TCR. We show that human H. saimiri-transformed T cell clones readily undergo cell death upon signaling via the TCR/CD3 complex or via phorbol 12-myristate 13-acetate (PMA) + ionomycin. The AICD in H. saimiri-transformed T cells was detectable a few hours after activation and it was not affected by the presence of interleukin (IL)-2 or by anti-CD4 cross-linking. However, AICD required tyrosine phosphorylation, since it could be blocked by herbimycin A. Cyclosporin A (CsA) did not block the development of AICD, but other consequences of activation in H. saimiri-transformed T cells like the production of interferon-γ. Surprisingly, the development of AICD was not reduced by neutralizing antibodies to tumor necrosis factor (TNF)-α or blocking antibodies directed to CD95 (Fas, APO-1), although H. saimiri-transformed T cells were sensitive to CD95 ligation. To confirm that this form of AICD is really independent of CD95, we have established an H. saimiri-transformed T cell line from a patient with a homozygous deletion in the CD95 gene. This CD95-deficient T cell line was as sensitive to AICD as other CD95-expressing H. saimiri-transformed T cells. In conclusion, we describe here a type of AICD in H. saimiri-transformed T cells that is independent of CD95 and TNF-α, not sensitive to CsA, but requires tyrosine phosphorylation. This system should be useful for the investigation of CD95-independent forms of AICD.     

The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae,ac-jun homologue,is involved in oxygen metabolism

Summary The PAR1/SNQ3 gene of S. cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box. The observed H₂O₂-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates. Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen. The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression. As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected. These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes. The finding that H₂O₂ promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.Dedicated to Professor Dr. R. W. Kaplan on the occasion of his 80th birthday     

IL-5 is essential for vaccine-induced protection and for resolution of primary infection in murine filariasis

The pathways conferring immunity to human filariases are not well known, in part because human-pathogenic filariae do not complete a full life cycle in laboratory mice. We have used the only fully permissive infection of mice with filariae, i.e., infection of BALB/c mice with the rodent filarial nematode Litomosoides sigmodontis. Our previous results showed that worm development is inversely correlated with Th2 cytokine production and eosinophilia. The scope of the present study was to directly elucidate the role of interleukin-5 (IL-5) and eosinophils in controlling the development of L. sigmodontis after vaccination and in primary infection. BALB/c mice immunized with irradiated third-stage larvae (L3) were confirmed to have elevated IL-5 levels as well as high subcutaneous eosinophilia and to attack and reduce incoming larvae within the first 2 days, resulting in 70% reduction of worm load. Treatment of vaccinated mice with anti-IL-5 antibody (TRFK-5) suppressed both blood and tissue eosinophilia and completely abolished protection. This demonstrates, for the first time in a fully permissive filarial infection, that IL-5 is essential for protection induced by irradiated L3 larvae. In contrast, in primary-infected mice, anti-IL-5 treatment did not modify filarial infection within the 1st month, most likely because during primary infection IL-5-dependent mechanisms such as subcutaneous eosinophilia are induced too late to disturb worm establishment. However, there is a role for IL-5 late in primary infection where neutrophil-dependent worm encapsulation is also under the control of IL-5. Received: 30 March 2000