On the origin of lateral asymmetry

Lateral asymmetry refers to unequal fluorescent intensity between adjacent regions of sister chromatids. It has been observed in the centromeric regions of mitotic chromosomes of mouse or human origin when cells are grown in 5-bromo-2-deoxyuridine (BrdU) for a single round of DNA synthesis. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used with pseudodiploid mouse cells to show that the regions of asymmetrical brightness coincide with major satellite repetitive DNA, and that the more heavily BrdU-substituted chromatid is the one that fluoresces less brightly. These observations support a 20 year old hypothesis on the origin of lateral asymmetry. Other observations suggest that differential loss of DNA from the heavily substituted chromatid also contributes to lateral asymmetry.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

After-Ripening in Festuca idahoensis Seeds: Adaptive Dormancy and Implications for Restoration

Festuca idahoensis (Idaho fescue) was a common native perennial bunchgrass in the sagebrush steppe of the western United States until the introductions of domestic livestock and alien plants. Restoration of Idaho fescue to degraded sites will likely involve reseeding, and one of the factors affecting reseeding success is germinability of the seeds employed. We investigated effects of after-ripening and storage temperature on germinability of Idaho fescue seeds collected from a central Oregon site. Six months of after-ripening were required before maximum germination was obtained. Storage of dry seeds at either room temperature (20°C) or at cooler, alternating temperatures (5/15°C) did not alter the rate at which dormancy was lost. Storage at the warmer temperature promoted rapid germination in seeds that had broken dormancy. Seed longevity varied greatly from year to year. Seeds produced in a very dry year had poorer germination and shorter longevity than seeds produced during a year with near normal precipitation. Because seed dispersal occurs in late July and early August for Idaho fescue in central Oregon, a six-month after-ripening requirement ensures that the greatest potential germination coincides with the spring period most likely to provide sufficient moisture for seedling establishment.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

Rapid and Sensitive Detection of Xanthomonas fragariae by Simple Alkaline DNA Extraction and the Polymerase Chain Reaction

Methods for DNA preparation from Xanthomonas fragariae in infected or artificially contaminated strawberry plants were compared in diagnostic assays using the polymerase chain reaction (PCR). The bacterium was detected using PCR with primers specific to a region of its hrp gene. Sensitivity of detection was 1.25 ×l 10³ CFU ml^-1 using DNA from bacterial suspensions prepared by an alkali extraction method. This was 10-fold more sensitive than DNA extraction by boiling, and was equal to that in which DNA was prepared by a more involved cetyltrimethylammonium bromide (CTAB) procedure. Sensitivity of detection from artificially contaminated strawberry tissues was 10-fold less than that from cell suspensions. The results indicated that a rapid and simple method of alkali DNA sample preparation is applicable for the sensitive and reliable detection of X. fragariae and possibly other plant pathogenic bacteria.     

Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R¹⁵⁵-S¹⁵⁶. Cleavage at R²⁷¹-T²⁷² generated fragment 1.2 and prethrombin 2 whilst cleavage at R²⁸⁴-T²⁸⁵ yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R³²⁰-I³²¹ which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1–3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R²⁷¹-S²⁷² and by α-thrombin at R¹⁵⁵-S¹⁵⁶ and R²⁸⁴-T²⁸⁵. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30–40 kDa. © 1995 Wiley-Liss, Inc.     

Effect of a membrane interactive peptide on plant cells of canola (Brassica napus) and two fungal pathogens

Summary A membrane interactive peptide was toxic to microspores, pollen and protoplasts of canola in the 1–5 µM concentration range. Similarly, at 5.0 µM the peptide completely inhibited germination of conidia ofVerticillium albo-atrum; however, when tested with conidia of a virulent isolate of blackleg (Leptosphaeria maculens), a fungal pathogen of canola, much higher levels (>30 µM) of the peptide were required to reduce or arrest germination and growth of the conidia. When testing the relative toxicities of novel peptides on plant cells and their pathogens, pollen germination is a simple, rapid and reliable alternative to protoplasts.     

Gypsy moth cell lines divergent in viral susceptibility

Summary A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus. This research was funded in part through a reimbursable agreement with the U.S. Forest Service.