Inhibition of human telomerase by a retrovirus expressing telomeric antisense RNA

Human telomerase, the RNA-dependent DNA polymerase that adds TTAGGG repeats to chromosome ends, is selectively expressed in immortalised cells and most tumours, suggesting a potential role for telomerase inhibitors in cancer therapy. Replication-deficient retroviruses were used to determine whether mRNA containing UUAGGG, the complementary sequence to the template region of the hTR telomerase RNA, is sufficient to inhibit telomerase activity. Telomerase activities measured by the telomeric repeat amplification protocol (TRAP) assay in extracts prepared from immortalised mouse fibroblasts, human HeLa cells and human kidney carcinoma cells were inhibited by 75% or greater in 26 of 56 cell clones expressing UUAGGG. Telomerase activity was not inhibited by expression of mRNA containing a transposed sequence, GGGAUU. Telomerase activities in vivo were inferred from changes in cellular morphology, proliferation capacity, growth rate and measurement of the content of telomere DNA. Giant senescent-like cells emerged shortly after cloning mouse PA317 and human HeLa cells expressing UUAGGG. The fraction of giant cells varied from 100% at the fifth population doubling (PD) in one culture to 2-6% at 50 PD in several other cultures. Giant cells were absent in all parental cells and clones expressing GGGAUU. The average cellular content of telomere DNA was independent of telomerase activity over 50 PD. The results indicate that expression of RNA complementary to the template region of hTR is sufficient to inhibit telomerase in vitro and in vivo, but that the effect of inhibition on individual cells is highly variable.     

Inhibition of eFGF expression in Xenopus embryos by antisense mRNA

This pilot study analyzed the bone reactions to early loaded titanium plasma-sprayed implants. A total of 24 titanium plasma-sprayed implants (12 in the maxilla and 12 in the mandible) (Primary Healing Implant, Legnano) were inserted into four Macaca fascicularis monkeys with instruments specially designed to obtain a precise fit of the implant in the bone socket. A metal superstructure was cemented into 10 mandibular and 10 maxillary implants 15 days after implant insertion. The four remaining implants were used as controls. Eight months after implant placement, a block section was carried out, the defect was filled with nonresorbable hydroxyapatite, and all 24 implants were retrieved. The implants were treated to obtain thin ground sections that were examined under normal and polarized light. Histologic analysis showed that bone was observed around the implant surface in all implants. Morphometric analysis demonstrated that bone lined 67.2% (SD = 3.1%) of the maxillary implant surface, and 80.71% (SD = 4.6%) of the mandibular implant surface. No differences were found in the percentage of bone-implant contact in the control implants. In the loaded implants, however, the bone around the implants had a more compact appearance. The study demonstrated that it is possible to obtain a high percentage of bone-implant contact in early loaded titanium plasma-sprayed implants.     

Studies on inhibition of activated oncogene expression by antisense RNA]

Blood group ABO antigens are known to be carried by several platelet glycoproteins (GP), e.g. GPIb, GPIIa, GPIIb, GPIIa and PECAM. Beside these proteins, we recently observed that blood group A antigen was also expressed on some other uncharacterized platelet proteins (70-90 kDa) having electrophoretic mobilities closely resembling those of GPIV and GPV. These findings prompted us further to characterize these latter ABO-expressing platelet proteins. By antigen capture ELISA, wherein the monoclonal antibodies (mAbs) CLB-IVC7 and CLB-SWI6 were used to hold the corresponding antigens GPIV and GPV, human anti-A specifically bound to these proteins derived from A1-platelets; neither GPIV nor GPV derived from A2-, B- or O-platelets bound anti-A. In a Western blot assay using immunoprecipitated GPIV and GPV as antigens, mAb anti-A immunostained GPIV and GPV precipitated from A1, but not from A2 and O platelets. These results conclusively demonstrate that blood group A antigen is expressed on platelet GPIV and GPV.     

Field study on the distribution of Entamoeba histolytica and Entamoeba dispar in the northern Philippines as detected by the polymerase chain reaction

The influence of the electron contamination at in vivo dosimetry with diodes on the patient surface has been investigated by introducing different accessories in the beam path and by changing the field size and SSD. The results show a clear correlation between the electron contamination at an effective measuring depth of the diode and the signal from the patient diode. When the electron contamination is taken into account the agreement between the diode values and the absorbed dose is greatly improved. More accurate in vivo dosimetry with less error margins is therefore possible if better predictions of the electron contamination in high-energy photon beams can be performed.     

Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR

Amoebiasis is caused by two distinct species, a pathogenic form (Entamoeba histolytica) and a nonpathogenic form (Entamoeba dispar), which are morphologically identical. Although the distinction between these two species is of great clinical importance, the methods developed for this purpose either are very time-consuming or involve laborious procedures for isolation of the DNA. We report here a simple PCR method starting with fresh stool specimen that allows for the sensitive and reliable distinction between E. histolytica and E. dispar. After initial concentration by the sodium acetate-acetic acid-formalin (SAF) method and digestion with proteinase K, a 0.88-kb sequence of the multicopy 16S rRNA gene served as a target for PCR amplification. The method starting with unpreserved specimens proved to be very sensitive and was not influenced by the quick exposure to SAF fixative during the initial concentration step. However, storage in SAF fixative prior to testing resulted in a decreased sensitivity within 2 days. The detection limit of the method was as low as one copy of the 16S rRNA gene. No cross-reactivity was observed with other common intestinal protozoa. Mixed infections involving both E. histolytica and E. dispar could easily be detected at a ratio of 1:10,000 by agarose gel electrophoresis or a DNA hybridization immunoassay.     

Inhibition of anchorage-independent growth of ras-transformed cells on polyHEMA surface by antisense oligodeoxynucleotides directed against K-ras

We examined the effect of antisense oligodeoxynucleotides (AS ODNs) directed against v-Ki-ras on the anchorage-independent growth of v-Ki-ras-transformed rat fibroblasts using plates coated with an antiadhesive polymer, poly(2-hydroxyethyl methacrylate) (polyHEMA). Among AS ODNs tested, 17mer AS ODN centered around codon 12 of v-Ki-ras inhibited the v-Ki-Ras expression and v-Ki-Ras-induced anchorage-independent growth, while its sense sequence did not affect it. Using polyHEMA plates, the direct addition of AS ODNs to the cells growing anchorage-independently and various treatment schedules of AS ODNs are now available to identify the most desirable conditions for AS ODNs treatment.     

Inhibition of host RNA polymerase II-dependent transcription by vesicular stomatitis virus results from inactivation of TFIID

The present study tested the hypothesis that one or more tyrosine kinase(s) are downstream of protein kinase C (PKC) in the signal transduction pathway responsible for the cardioprotective effect of ischemic preconditioning (PC). Isolated rabbit hearts were subjected to 30 min of regional ischemia followed by 2 h of reperfusion. Infarct size was measured by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarction in control hearts was 32.9+/-1.8%. Ischemic PC with 5-min ischemia/10-min reperfusion reduced infarct size to 11.5+/-1.5% (P<0.05). Infusion of the tyrosine kinase inhibitors, genistein (50 microM) or lavendustin A (0.5 microM), alone did not affect the level of infarction. When infused around the 5-min PC ischemia genistein failed to block protection (13.7+/-1.0%). However, when present at the onset of the 30-min ischemia both genistein and lavendustin A completely aborted protection (31.4+/-2.0 and 28.1+/-1.5%, respectively). Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 0.05 nmol) was as protective is ischemic PC (14.9+/-3.0%; P<0. 05). Similar to PC, PMA-induced protection was completely prevented by both genistein and lavendustin A. Conversely, anisomycin (50 ng/ml), an activator of MAP kinase kinases (dual tyrosine and threonine kinases), was very protective (7.5+/-1.6%; P<0.05) and this protection was still present when PKC was inhibited by 5 microM chelerythrine (12.1+/-1.6%; P<0.05). In conclusion, activation of a tyrosine kinase during the long ischemia appears to be required for cardioprotection in the rabbit heart. Furthermore, the ability of tyrosine kinase inhibitors to block PMA-induced protection in conjunction with the failure of PKC inhibition to prevent anisomycin-induced protection suggests that the tyrosine kinase is downstream of PKC and that the tyrosine kinase may be a MAP kinase kinase.