The ROOT HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement in Arabidopsis.

In plants, morphogenesis is largely determined by the orientation and extent of cell enlargement. To define the molecular mechanisms regulating plant cell enlargement, we have conducted a molecular genetic analysis of the ROOT HAIR DEFECTIVE3 (RHD3) gene of Arabidopsis thaliana. Mutations affecting the RHD3 gene were found to alter cell size, but not cell number, in tissues throughout the plant. Genetic and physiological analyses suggest that the RHD3 gene is not required for proper cell type specification, and it is likely to act downstream of the hormones auxin and ethylene. The RHD3 gene was cloned by a T-DNA tagging method and confirmed by the molecular complementation of the rhd3 mutant phenotype and by the analyses of six rhd3 mutant alleles. Consistent with the global effects of the rhd3 mutations, the RHD3 gene is expressed in all major plant organs. The deduced RHD3 product is a novel 89-kD polypeptide with putative GTP-binding motifs near the amino terminus. RHD3-like genes were identified from a protozoan (Entamoeba histolytica), a fungus (Saccharomyces cerevisiae), and another plant species (Oryza sativa), with the sequence identity including the putative GTP-binding motifs. These results imply that the RHD3 protein is a member of a new class of GTP-binding proteins that is widespread in eukaryotes and required for regulated cell enlargement.

Ethanol induced morphologic alterations during growth and maturation of cardiac myocytes.

Alteration of growth and development of cells exposed to ethanol during embryogenesis contributes to dysmorphism. The mechanism(s) of these alterations remains an enigma. This paper describes studies of an in vitro cardiac myocyte model in which the major effort was to investigate growth and development parameters in an obligate interacting multicellular system. The well defined events of in vitro myogenesis allow for documentation or dysgenesis and altered growth in the presence of the taratogen, ethanol. The cells exposed to ethanol did not mature morphologically or functionally compared with controls. Increasing concentrations of ethanol appear to have a graded damaging effect. The greater the concentration of ethanol the more profound the dyssynchronous growth. The morphologic correlates were multinucleation, and alteration in the ultrastructural organization of cell-cell contacts and myofilaments. Correlation of these findings with those observed in dysgenic muscle of human infants and rat pups exposed to ethanol in utero, may provide at least a partial understanding of the teratogenic manifestation of ethanol in embryogenesis and organogenesis.

Closed chamber system for delivery of ethanol to cell cultures.

The accuracy and consistency of the delivery of ethanol to cultured cells is important to determine effects on morphologic, biochemical and physiologic alterations. Open and closed chamber systems were evaluated to determine cytotoxic vs sublethal, potentially teratogenic effects on neonatal rat cardiac myocytes. The open system employed a variety of cell culture vessels. Cardiac cells were exposed directly to ethanol in the growth media at concentrations of 5-50 mM in Petri dishes, multiwell slides and multiwell chambers. Ethanol concentrations in the media in these open vessels decreased over 60% in a 24 hr incubation period. A closed system consisted of tightly sealed plastic containers in which the same vessels were used. The vessels were placed on a platform over a bath of ethanol-water. Cells were acclimated for 24 hr with ethanol in the bath at 200% of the final desired media concentration. Ethanol gradually diffused into the media to reach peak levels of 5, 10, 25 or 50 mM at 24 hr. After the 24 hr period, ethanol was added to both the media and bath at the desired concentration. Cells exposed gradually to ethanol in the closed chambers remained viable, but showed slower division and growth. A period of gradual acclimation is required to induce sublethal cellular effects rather than lethal effects. The diversity of cell systems and manipulations of cultures to study the potential teratogenic effects of ethanol are improved using such a closed chamber system.