Universal primers for amplification of three non-coding regions of chloroplast DNA

Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants.     

Measles-specific T cell clones derived from a twin with multiple sclerosis: genetic restriction studies

The association between multiple sclerosis (MS) and HLA-DR2 suggests that the disease may be associated with an aberrant immune response, likely directed against an antigen of either viral or host origin. We have used measles virus-specific T cell clones derived from a patient with MS to study genetic restriction patterns of antigen presentation by macrophage-enriched (E-) populations. Twenty-two clones proliferated in response to measles-infected Vero cells but not to mumps-infected or uninfected Veros. E- cells from both the autologous subject and her healthy, measles nonresponder identical twin were capable of presenting antigen to all clones. Studies with E- cells obtained from a panel of cell donors demonstrated clones which recognized antigen in association with D2/DR2, DR4, subgroups of DR4, and SB3. Three clones recognized antigen only in association with the autologous or twin's cells, but not with other sets of HLA-matched E-cells obtained from healthy donors or from other patients with MS. These studies indicate that the differing responses to measles virus demonstrated by these two identical twins are not explained by alterations in the interactions between antigen-presenting cells and T cells. Furthermore, at the clonal level, no preferential role is seen for HLA-DR2 as the restricting element for presentation of measles virus to these clones.     

Cyclic AMP treatment of Rous sarcoma virus-transformed Chinese hamster ovary cells increases phosphorylation of pp60src and increases pp60src kinase activity

Treatment of growing Rous sarcoma virus-transformed Chinese hamster ovary cells with the cyclic AMP analog 8-bromo-cyclic adenosine 3',5'-monophosphate (8-bromo-cyclic AMP) stimulates the incorporation of 32Pi into the viral transforming protein pp60src. Based on one-dimensional and two-dimensional peptide analysis and phosphoamino acid analysis, the increase is on a single phosphoserine residue at the NH2 terminus of the protein. The phosphate incorporation increases during the first 4 h of treatment. The pp60src kinase activity in extracts of cells treated with 8-bromo-cyclic AMP was stimulated about 2- to 3-fold. This stimulation of kinase activity increased during the first 3 h of treatment with 1 mM 8-bromo-cAMP and the activity was increased in both the soluble and particulate fraction of the cells. These results suggest that cyclic AMP can modulate the activity of pp60src in transformed cells.     

Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor

We have identified a tyrosine kinase activity present in tumors which were raised in rats by subcutaneous injection of Rous sarcoma virus-transformed rat cells (SR-NRK). This kinase phosphorylates tyrosine on the heavy chain of IgG from tumor-bearing rabbit (TBR) sera specific for the src gene product, pp60src. Using TBR-IgG phosphorylation as an assay, we have purified this kinase over 7200-fold. The purification procedure involves detergent extraction of tumors followed by sequential column chromatography on hydroxylapatite, DEAE-Sephacel, oligodeoxyadenosine-cellulose, an affinity column prepared from TBR-sera, and Sephacryl S-200. The IgG kinase activity behaves as a molecule of apparent Mr = 54,000 on Sephacryl S-200 molecular sieve chromatography. Analysis of the Sephacryl fractions by SDS-PAGE indicates that a major Coomassie blue-stained band with an apparent Mr = 54,000 (p54), co-elutes with the peak of kinase activity. From 600 g of tumors, approximately 200 micrograms of p54 are obtained. We have four types of evidence which show that p54 is related to pp60src. 1) Purified p54 is capable of undergoing endogenous phosphorylation in the presence of [gamma-32P]ATP producing a 32P-labeled pp54 polypeptide which is specifically immunoprecipitated by TBR-sera and contains only phosphotyrosine. 2) Purified p54 competes with 32P-labeled pp60src for binding to TBR-IgG, indicating a degree of purification over starting material which agrees very well with the results obtained by the IgG kinase assay. 3) V8 protease digestion of pp60src and p54 suggests that they share a common 26,000 fragment. 4) Antibodies to partially purified p54 specifically precipitate pp60src from Rous sarcoma virus-transformed chicken cells.     

Steroids and human breast cancer.

Normal mammary gland cells are sensitive to a number of hormones, of which estrogen and prolactin exert the most obvious effects. Some breast cancer cells are also sensitive. Cytoplasmic receptor sites for each hormone are responsible for the interaction between the hormone and the cell. The presence of estrogen receptor has been especially studied in humans. Data collected from several sources are reviewed. The prese nce of estrogen receptors has been assayed in 154 primary breast tumors and 72 metastatic breast tumors for correlation with response to endocri ne therapy. Positive values were found in 70% of primary and 58% of metastatic specimens. Of 211 treatment trials, ablative therapy produced objective tumor regressions in 33%. Of the 94 trials with negative receptor values, only 8 were successful while 59 of the 107 trials in patients with positive receptor values succeeded. In those with borderline tumor receptor, values had a 30% response. With additive therapy, 34% of 170 trials showed tumor regression. Of these, 82 had negat ive receptor values but 8% were successful, whereas of 85 with positive receptor values, 60% were favorable. With miscellaneous therapy, 27% of 55 trials gave responses to a variety of endocrine therapies, including antiestrogens. The 32 with negative receptor values gave 16% of favorable responses whereas 43% of 23 trials in those with positive receptor values succeeded. Estrogen receptor assays performed routinely would spare patients with negative results from unnecessary major ablative therapy. Of those with positive findings, 55-60% might be benefited. The fact that all with positive receptor values do not respond is attributed to the fact that this is only part of the hormonal control system. Other biochemical lesions are assumed to have occurred in patients when endocrine therapy fails despite positive estrogen receptor levels as measured.     

Ecological specialization and niche overlap of subterranean rodents inferred from DNA metabarcoding diet analysis

Knowledge of how animal species use food resources available in the environment can increase our understanding of many ecological processes. However, obtaining this information using traditional methods is difficult for species feeding on a large variety of food items in highly diverse environments. We amplified the DNA of plants for 306 scat and 40 soil samples, and applied an environmental DNA metabarcoding approach to investigate food preferences, degree of diet specialization and diet overlap of seven herbivore rodent species of the genus Ctenomys distributed in southern and midwestern Brazil. The metabarcoding approach revealed that these species consume more than 60% of the plant families recovered in soil samples, indicating generalist feeding habits of ctenomyids. The family Poaceae was the most common food resource retrieved in scats of all species as well in soil samples. Niche overlap analysis indicated high overlap in the plant families and molecular operational taxonomic units consumed, mainly among the southern species. Interspecific differences in diet composition were influenced, among other factors, by the availability of resources in the environment. In addition, our results provide support for the hypothesis that the allopatric distributions of ctenomyids allow them to exploit the same range of resources when available, possibly because of the absence of interspecific competition.