Role of svp in Drosophila pericardial cell growth.

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DAI muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.

[Establishment and application of double color and fiber-FISH].

To determine whether genetic material in 5q13.3 breakpoint region changed in the course of establishing the cell line with a constitutional chromosomal inversion inv(5)(p13.1q13.3) associated with hairy cell leukemia, double color fluorescence in situ hybridization (FISH) on metaphase, interphase and DNA fibers were performed in cells of the cell line with the cosmids cCI5-216 and cCI5-267 DNA probes labeled by either biotinylate or digoxigenin. It showed that the cell line gave the same results as those of the original cells, which indicated that no change of the genetic material at 5q13.3 breakpoint region occurred. The cell line is valuable to reveal the molecular pathogenesis of hairy cell leukemia.

[Functional analysis of the human T-type calcium channel alpha 1H subunit gene in cellular proliferation].

An increase in the functional expression of the T-type calcium (Ca) channel previously has been proposed to be associated with vascular smooth muscle cell proliferation although direct evidence for the channel causing these effects remains to be demonstrated. In this study, we provide evidence that stable over-expression of the alpha 1H subunit of the T-type Ca channel (CACNA1H) in HEK-293 cells confers a significant growth advantage. Over-expression of the alpha 1H subunit of the T-channel was confirmed in stable transfects by RT-PCR analysis using specific primer pairs and also by electrophysiology. Growth curve assays showed population doubling time for alpha 1H stable transfects was 14.0 +/- 0.4 h, whereas control cultures had a population doubling time of 22.1 +/- 1.1 h (n = 3, P < 0.05). In addition, total cell numbers significantly increased in stable transfects at all time points investigated from 48-120 h after plating (5 x 10(3) cells/well) compared with control cultures. Consistent with these findings flow cytometry showed that 53.9% of control cells were in G1, 34.5% in S and 11.6% in G2/M whereas alpha 1H transfectants displayed 45.6% of cells in G1, 44.6% in S and 9.8% in G2/M. Finally, the Western blotting results verified that the levels of protein expression of CDK2, cyclin A and cyclin E were relatively high in alpha 1H transfectants compared to control cultures. Our results demonstrate that the T-type Ca channel provides a growth advantage to HEK-293 cells when stably expressed and that it probably exerts these effects via control of the G1/S cell cycle mechanism.

[The use of RNAi as a technique to study the functions of heart-related genes in Drosophila].

RNAi is a recently developed method to block the activity of cellular genes by artificially providing sense and anti-sense RNA corresponding to a target gene. By inducing rapid degradation of the corresponding endogenous mRNA and blocking new mRNA synthesis, RNAi leads to post-transcriptional gene silencing. Now this phenomenon has been claimed to exist in C. elegans, Drosophila, buds, fungi and plants and is being used to study the functions of some special genes or the known genes at specific time point. It is extremely useful for those genes or organisms that their mutants are not easily obtained. The Drosophila heart related genes, tinman and wingless, have been shown to play an important role in coordinating the early formation of heart progenitor cells and precursors, yet the late function is still unexplored. In this experiment, we took the advantage of RNAi technique, microinjected tinman and wingless dsRNA into the early embryos in Drosophila respectively and got these two genes' RNAi phenotypes, which were very similar to that of their mutant, showing heart tube defects or no heart precursors formation. tinman dsRNA even caused visceral mesoderm defects and the somatic muscles disruption, yet wingless dsRNA only affected heart precursors and had no effect on visceral mesoderm and somatic muscles, indication that the heart-related genes dsRNA interference worked effectively and exclusively in Drosophila.