Methods for study of mutations and mutagenesis in human lymphocytes

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.     

SCENEDESMUS WALL ORNAMENTATION. I. SCENEDESMUS PARISIENSIS CULTURES1

Four strains of Scenedesmus parisiensis Chodat were studied in xenic and axenic culture in 3 media as well as in cultures incubated in sterile vessels in nature. Organized coenobia are usually produced but these may have merely short spines, spines and serrate edges, or lack wall ornamentation. Because the serrate edge is either not formed or cannot be readily detected in most cases, it is not a satisfactory morphological feature for delimiting this species. In laboratory studies it is noted that S. parisiensis might be confused with S. denticulatus rather than S. brasilien-sis. Inasmuch as both xenic and axenic cultures of the -f strains produced similar results, S. parisiensis can be readily characterized.     

Diffusion effects in calcium-regulated strain fields

The Goodwin-Trainor equations for cellular morphogenesis, based on calcium ion regulation of the visco-elastic properties of the cellular cortex, are generalized to the situation where the concentration of calcium ions (free plus bound) is allowed to change locally. A stability analysis is presented which shows that the generalized equations are also stable against perturbations at low and high wave lengths and may, for certain parameter values, develop instabilities at intermediate wave lengths.     

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.     

Growth of Daphnia magna in the laboratory in relation to the nutritional state of its food species, Chlamydomonas reinhardtii

Growth rates and fecundities of Daphnia magna in the laboratorywere higher, and mortalities were lower, when the animals werefed on log-phase cells of Chlamydomonas reinhardtii than onnitrogen- or phosphorus-limited cells. The effect appears tobe related to the nutritional adequacy of the algae, ratherthan to their production of toxic or inhibitory substances,but it was not related directly to their nitrogen content orgrowth rates.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.