Evaluation of alpha-Tubulin as an Antigenic and Molecular Probe to Detect Giardia lamblia

The alpha/beta-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant alpha-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of alpha-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the alpha-tubulin gene from G. lamblia. PCR-RFLP analysis of this alpha-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that alpha-tubulin can be used as a molecular probe to detect G. lamblia.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II

Abstract: One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment.A promising method to do this is by use of pathogen-specific molecular probes. However,this approach has been little used to date.We constructed,and validated in the laboratory,a fluoro-chrome-labeled molecular probe specific to Aurantimonas coralicida ,the bacterial pathogen of the Caribbean coral disease white plague type II (WPII).We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen.Probe design was based on a unique subset (25 nucleotides)of the complete16S rRNA gene sequence derived from a pure culture of the pathogen.The pathogen-specific probe was labeled with the fluorochrome GreenStar*™FITC (fluorescein isothiocyanate,GeneDetect Ltd,New Zealand).As a control, we used the universal eubacterial probe EUB 338,labeled with a different fluorochrome (TRITC,tetra-methyl-rhodamine isothiocyanate).Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II),healthy controls,and corals with an uncharacterized disease ("patchy necrosis ").All samples were analyzed using fluorescence in situ hybridization (FISH).We have determined that the probe is specific to our laboratory culture of the coral pathogen,and does not react with other bacterial species (the eubacterial probe does).The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n=9 samples)exhibiting signs of both WPI and WPII.Diseased (and healthy)tissue samples (n=4)from corals exhibiting signs of "patchy necrosis "were also assayed.In this case the results were negative, indicating that the same pathogen is not involved in the two diseases.Incorporation and use of pathogen-specific probes can...

Analysis of p53 tumor suppressor gene mutations and human papillomavirus infection in human bladder cancers

To determine whether the dysfunction of p53 caused either by mutation of the p53 gene itself or by binding to E6 protein of oncogenic HPVs is involved in the transitional cell carcinomas (TCCs) of the bladder, we analyzed 23 TCCs of the bladder. DNA was extracted from each paraffin embedded tissue of TCCs of bladder and polymerase chain reaction (PCR)/single strand conformation polymorphism (SSCP) analysis were performed to screen mutations in p53 tumor suppressor gene, then PCR/dot blot hybridization were performed to detect infection of HPVs. We found that p53 gene mutation was found in 3 cases and oncogenic HPV infection was detected in 8 cases and thus, the overall incidence of possible p53 dysfunction was 47.8% on DNA analysis (If the results of immunohistochemistry to detect overexpression of p53 protein were included, the incidence was 60.9%). Therefore, we concluded that dysfunction of p53 plays a major role in the development of TCCs of bladder in Korean patients.

Prenatal fetal sex determination from maternal peripheral blood using polymerase chain reaction

We have investigated the use of a nested polymerase chain reaction(PCR) assay with Y-specific sequence from the DYS 14 locus on the short arm of Y-chromosome for prenatal sex determination in the peripheral blood of 22 pregnant women who participated in the antenatal genetic diagnosis program. The sensitivity and specificity of the nested PCR using DYS 14 locus primers(Y1.5,Y1.6, and Y1.7,Y1.8) were 76.4% and 55.5%, respectively. In terms of gestational age, positive predictive values of 66.6%, 66.6%, and 80% were obtained for the first, second, and third trimester respectively. The corresponding negative predictive values were 50%, 50%, and 100% respectively. Male specific band was positive in three of the six cases of female bearing women and male specific band was negative in three of the seven cases of male bearing women during 9-16 gestational weeks showing low sensitivity. But all cases except one show the male specific band during the male fetus and all female fetuses did not show the male specific 198 base pair band during 18 approximately 40 gestational weeks. This study suggests that prenatal sex determination by PCR employing maternal peripheral blood was usually possible in late pregnancy but less reliable in early pregnancy. It seems that if we used a method separating fetal cells from maternal blood and then run PCR on these cells with DYS 14 locus primers we could make a fairly accurate fetal sex determination.

Mutations in the pre-core region of hepatitis B virus DNA in patients with chronic liver diseases

To investigate the prevalence of point mutation in the pre-core (pre-C) region of hepatitis B virus (HBV) DNA, we performed dot blot hybridization and sequencing of enzymatically amplified HBV DNA from the sera of 25 patients with HBeAg-positive and 32 patients with HBeAg-negative chronic liver diseases. The pre-C region of HBV DNA was successfully amplified by polymerase chain reaction (PCR) from 55 (96.5%) of 57 sera. According to the status of serum HBeAg, HBV DNA was amplified from all 25 sera of HBeAg-positive patients and 30 (93.8%) of 32 sera of HBeAg-negative patients. All amplified DNA from the sera of 25 patients with HBeAg-positive and that from 28 (93.3%) of 30 patients with HBeAg-negative chronic liver diseases hybridized with the wild type probe. In addition, that from 5 (20.0%) among 25 patients with HBeAg-positive and 16 (53.3%) among 30 patients with HBeAg-negative chronic liver diseases hybridized also with the mutant type probe. These results suggest that the prevalence of point mutation in the pre-C region of HBV DNA is relatively high in patients with HBeAg-negative chronic liver diseases and further study is mandatory to identify the significance of this mutation.

Nucleotide sequence analysis of HTLV-I isolate from a Korean patient with HAM/TSP

Limited nucleotide sequences of human T-cell lymphotropic virus type I (HTLV-1) provirus isolated from the first case of a Korean patient with HTLV-I associated myelopathy and tropical spastic paraparesis (HAM/TSP) were analysed and compared with other isolates from different regions of the world. The sequences of the env, LTR regions (536bp, 690bp respectively) showed 98.7%, 99.3% homologies with the prototype HTLV-I, ATK-1, isolated from a Japanese Adult T-cell leukemia (ATL) patient. A comparison between other isolates from different geographical origins revealed that the Korean HTLV-I isolate is more closely related to Japanese isolates than to those from other geographical origins