Dose responses for chromosome aberrations produced in noncycling primary human fibroblasts by alpha particles,and by gamma rays delivered at sublimiting low dose rates

As the total dose of X or gamma rays is delivered at lower and lower rates, the yield of chromosome aberrations progressively diminishes. Simultaneously, the shape of the dose response changes from one exhibiting pronounced upward curvature at high dose rates to one approaching linearity at low dose rates. Although the maximum sparing effect caused by lowering the dose rate can be predicted from classical cytogenetic theory, it has yet to be verified experimentally. Here, noncycling normal human fibroblasts were exposed to graded doses of (137)Cs gamma rays at chronic dose rates of 6.3 and 2.8 cGy h(-1), dose rates that we reasoned should be lower than those required to achieve maximal sparing. This was indeed shown to be the case, after it was determined that the two chronic dose rates produced identical linear dose responses of 0.05 total aberrations per cell Gy(-1). Consistent with cytogenetic theory, this value was statistically indistinguishable from the linear coefficient derived from a fit to aberration frequencies produced by high-dose-rate exposure. Exposure to (238)Pu alpha particles also produced a linear dose response for total aberrations, whose slope-with respect to (137)Cs gamma rays as a reference radiation-implied a maximum RBE of 35 +/- 2.     

A rapid selection/amplification procedure for high-level expression of recombinant protein in a metal-amplifiable mammalian expression system

We describe a strategy for the selection and amplification of foreign gene expression in Chinese hamster ovary (CHO) cells employing a metallothionein gene-containing expression vector. This report describes an amplification procedure that results in an enrichment of clones exhibiting high levels of recombinant protein production and reduces the labour required for screening recombinant cell lines.     

Natural taxonomy of collagen based on amino acid composition

Hierarchical cluster analysis and principal components have been used to provide a more detailed separation of the collagens into natural taxonomic groupings than previously obtained. These groups strongly reflect the evolutionary development of collagen. The first component separates land- from sea-based animals, primarily based on the hydroxylation of lysine and proline, indicating that control of hydroxylation, a post-translational event, has exerted a dominant influence during evolutionary adaptation. The power of the technique is illustrated by the ability to partially separate the evolutionarily closely related main homothermic species. Furthermore, the genetically different fibrous collagens, Types I and III, are well separated from basement membrane Type IV collagen and the filamentous collagens. The technique could, therefore, in addition to providing a taxonomic grouping, classify any new collagen and provide clues to its evolutionary development.     

Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa

Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.     

Genotyping the baboon ABO histo-blood group locus by two-color fluorescence SSCP

The use of baboons in experimental medicine, especially as organ and tissue donors, would be facilitated by the availability of ABO histo-blood group O animals, which are currently rare. To meet our need in breeding and identifying such animals, we have developed a single-stranded conformational polymorphism (SSCP)-based genotyping assay using fluorescence detection. Various buffers and labeling schemes were evaluated to optimize allele discrimination. Through the use of a nested PCR protocol, single-cell sensitivity was achieved, making the assay applicable to preimplantation diagnosis following in vitro fertilization. We discuss the comparative advantages of SSCP vs. alternative methodologies for genotyping.     

High frequency of polyandry in a lek mating system

The adaptive significance of polyandry by female birds in theabsence of direct benefits remains unclear. We determined thefrequencies of polyandrous mating and multiple paternity inthe ruff, a lekking shorebird with a genetic dimorphism inmale mating behavior. More than half of female ruffs mate with, and have clutches fertilized by, more than one male. Individualfemales mate with males of both behavioral morphs more oftenthan expected. Polyandrous mating was more likely followingcopulation interference, but interference was uncommon. Themultiple paternity rate of ruffs is the highest known for avian lekking species and for shorebirds. The general hypothesis thatpair-bond constraints are the major selective factor favoringmultiple mating in birds does not predict our findings. Activegenetic diversification, which has been widely dismissed asa functional explanation for polyandrous mating in birds, mayapply with respect to the behavioral polymorphism in ruffs becauseof a Mendelian genetic basis for male behavioral morph determinationand aspects of male—male cooperation and female choice.However, rates of multiple paternity in other species of lekkingbirds are higher than generally realized, and the potentialbenefits of diversification in general deserve further consideration.