Transcription analysis of TIMP-1 and NM23-h1 genes in glioma cell invasion

PURPOSE: To evaluate using transcription analysis the presence and importance of two genes: NM23-H1 and TIMP-1 on control of tumor cell invasion in diffuse astrocytomas (WHO II) and glioblastoma multiforme (WHO IV). METHOD: Northern blot analysis of NM23-H1 and TIMP-1 was performed. Eight diffuse astrocytomas and 19 glioblastomas (WHO IV) were analyzed to determine if TIMP-1 and NM23-H1 were candidates to inhibition of tumor cell invasion quantitated RNA levels. The samples were collected directly from operating room. Total cellular RNA was extracted from frozen tissue samples using guanidinium-isothiocyanate and cesium chloride gradients. Total RNA (10 mg per sample) from tumor tissue were size fractionated through 1% agarose-formaldehyde gel and transferred to nylon filters and then hybridized to 32P-labeled DNA probes and placed for autoradiography. Levels of specific RNAs were determined by computer-assisted laser densitometry. Blot filters were sequentially hybridized to nm23 and TIMP-1 probes in addition to GAPDH, as a control. Statistical analyses were carried out according to t-test for equality of means. RESULTS: NM23-H1 were detected in each sample, however it did not correlate with malignancy and invasiveness. On the other side TIMP-1 gene expression showed a clear correlation between low expression and invasiveness. CONCLUSION: The data suggest that TIMP-1 is an inhibitor of high grade gliomas invasion. NM23-H1 was present in the entire gliomas sample, but it did not vary in diffuse astrocytomas and glioblastomas.

Gamma-radiation induced agglomeration of chicken muscle myosin and actine

Radiolytic behaviour of themajor vertebrate muscle proteins: fibrillarmyosin (molar mass, Mm = 520,000 g/mol) and filament forming actin (Mm = 42,050 g/mol) was studied using a SDS-polyacrylamide gel electrophoresis and quantified by high precision laser-densitometry. In order to study the OH radical contribution to the radiation damage, purified chicken myosin and actin (4 ?M) were prepared in N2O saturated solution and irradiated with 1?3 kGy at 60Co gamma source. With respect to changes in the molecular mass, the only observed myosin and actin damage was dose dependent agglomeration of proteins. The corresponding radiation chemical yields of 5 x?10-8 mol J-1 and 6.3 x?10-8 mol J-1 were obtained for myosin and actin, respectively. This result confirmed that only the radiation-induced agglomeration is initiated with the reaction of the OH radical even in the situation where the OH radical concentration produced exceeds the protein concentration 500 times thus enabling the multi-radical attack to occur.

Screening fetal losses for monosomy X with a simple PCR-based procedure

To screen for monosomy X in spontaneous fetal losses we explored a simple molecular strategy based on loss of heterozygosity at highly polymorphic X-linked loci. We developed a multiplex fluorescent procedure that allows the simultaneous amplification of five dinucleotide repeat polymorphisms in a large low-recombination region in the long arm of the X chromosome. Analysis was performed by computer-assisted laser densitometry. We did not find any instances of homozygosity at all five loci in 30 normal females tested, nor among 37 women whose typing data were retrieved from the Fondation Jean Dausset - CEPH genotype database. In addition, all cases of monosomy X previously diagnosed by conventional cytogenetics presented the anticipated loss of heterozygosity at all loci. We studied 19 spontaneously aborted female fetuses and we found four samples homozygous for the five loci (21%), in good agreement with the expected rate of monosomy X in first trimester spontaneous abortions. We conclude that the loci have high diversity and high efficiency in PCR-amplification and that our multiplex procedure constitutes a simple and useful molecular screening test for monosomy X in abortions and stillbirths.