[Stable transfection of DAOY cells with a GM3 synthase antisense construct and transient reduction in ganglioside content].

Tumor cell gangliosides are bioactive molecules involved in tumor-host interactions. To investigate their role in tumor formation and angiogenesis, we sought to develop an inhibitory model targeting human GM3 synthase, an essential enzyme in the ganglioside synthesis pathway, by antisense transfection. We prepared a number of transfectants from DAOY human medulloblastoma cells and isolated clones that stably expressed a 560-bp fragment of human GM3 synthase cDNA, in either sense or antisense orientation, as well as clones transfected with an empty vector. Both sense and antisense clones permanently incorporated mammalian expression vectors into their genomes. The DAOY cell clones were screened for ganglioside content using total lipid extraction, ganglioside isolation, and HPTLC. One antisense-transfected clone, 7.2A, in which total ganglioside content was reduced by 70%, was selected for further study. All sense- and sham-transfectants had ganglioside levels not different from that of untransfected DAOY cells. After 10 passages however, while antisense mRNA expression was fully maintained, the ganglioside content of 7.2A cells had reverted to normal levels. Antisense RNA transfection can sometimes have a reversible effect on the expression of a target. Possible regulatory mechanisms of this previously unrecognized process of reversion to wild type phenotype are discussed.

[Time-resolved immunofluorometric assay for DNA and DNA level in human serum]. / Vremiarazreshennyi immunofluorestsentnyi analiz na DNK i issledovanie soderzhaniia DNA v syvorotke cheloveka.

It is widely accepted that normal levels of extracellular DNA in the blood are quite low and can be elevated in systemic lupus erythematosus, some cancers, and after irradiation. The goal of this work was the development of a quantitative assay for DNA measurement in serum of healthy donors as well as in patients. Several approaches to IgG antibody induction against native mammalian DNA were tested. Antiserum containing such antibodies was obtained following immunization of rabbits with denatured DNA complexed to methylated BSA (in one of six animals). A time-resolved immunofluorimetric sandwich assay for DNA was developed using the rabbit antibodies. Sensitivity of the assay is 8 pcg/ml and the range 10-100,0000 pcg/ml DNA. The assay was employed for determination of DNA serum content in normals, viral hepatitis patients, and autoimmune thyroiditis patients. Serum DNA concentration in 38 healthy males was 0.62 +/- 0.49 ng/ml (medium +/- SD), as opposed to 2.3 +/- 6.6 [symbol: see text] 105 +/- 175 ng/ml in 19 hepatitis (B and C) patients and 14 autoimmune thyroiditis patients respectively. The high levels of extracellular DNA in circulation may contribute to induction of DNA-hydrolyzing antibodies often found in these disorders. The assay can also be used for measurement of low DNA concentrations against high protein background in other biological fluids.

Sources of incongruence among mammalian mitochondrial sequences: COII, COIII, and ND6 genes are main contributors.

To investigate the origins of incongruence among mammalian mitochondrial protein-coding genes, we compiled a matrix that included 13 protein-coding-genes for 41 mammals from 14 different orders. This matrix was examined for congruence using different partitioning strategies. The incongruence length difference test showed significant incongruence among the 13 gene partitions used simultaneously, and the result was not affected by third codon or transversion weighting. In the pair-wise comparisons, significant incongruence was detected between NADH:ubiquinone oxidoreductase subunit 6 gene (ND6), cytochrome oxidase subunit II (COII), or cytochrome oxidase subunit III (COIII) gene partitioned individually against the rest of the genes. Omission of any of the 14 mammalian orders alone or in combinations from the matrix did not result in a statistically significant improvement of congruence, suggesting that taxonomic sampling will not improve congruence among the data sets. However, omission of the ND6, COII, and COIII significantly improved congruence in our data matrix. Possible origins of unusual phylogenetic properties of the three genes are discussed.

[Development of a two-site immunoassay using polyclonal antibodies for determination of lactoferrin in human blood]. / Razrabotka dvukhsaitnogo immunoanaliza s ispol'zovaniem poliklonal'nykh antitel dlia opredeleniia laktoferrina v syvorotke krovi cheloveka.

The two-site enzyme-linked immunosorbent assay (ELISA) for lactoferrin using polyclonal antibodies to spatially distant epitopes has been developed. The assay sensitivity defined as minimal detectable lactoferrin concentration for p = 0.05 is 0.5 ng/ml. Accuracy of the assay (variance coefficient) is 7% within the clinical range of antigen concentrations. Human albumin, hemoglobin, and transferrin in concentrations up to 5 mg/ml practically do not interfere with the measurement. Sera of healthy donors and viral hepatitis patients were investigated using the two-site ELISA. The lactoferrin content in 44 donors' sera was 130 +/- 40 ng/ml (medium +/- standard deviation). A study of the serum specimens of 95 patients with hepatitis A, B, and C showed significant increase in serum lactoferrin concentration: 850 +/- 420, 780 +/- 580, and 680 +/- 500 ng/ml respectively. The assay showed good characteristics and may be recommended for lactoferrin measurement in patients' sera.