A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.     

Factors influencing species diversity in saline waters of Death Valley,USA

Salinity is a major factor influencing the distributions and abundances of aquatic macroinvertebrates of saline waters in Death Valley, California, USA. A general pattern of declining numbers of species with increasing salinity is seen in Death Valley waters. Some species are restricted to low salinities, others are found only in highly saline pools, and still others are widely distributed over a broad range of salinities.Salinity alone cannot explain distributions seen in the field. Distributions and abundances of species such as the caddisfly Limnephilus assimilis Banks are broader than would be predicted on the basis of laboratory studies of salinity and temperature. I present evidence that for such species, biotic factors such as reduced predation at high salinities may compensate for increased physiological stress.     

Interactions of sulfated mucopolysaccharides with lectins. Application to the separation of mucopolysaccharide mixtures.

The interaction of sulfated mucopolysaccharides and lectins has been studied by determining the amount of precipitate formed when mucopolysaccharides are added to a solution of concanavalin A or a partially purified lectin preparation from red kidney bean (Phaseolus vulgaris). The amount of insoluble complex obtained when a given mucopolysaccharide is added to a solution of partially purified red kidney bean preparation is pH dependent. The reaction of concanavalin A and heparin has also been studied by adding increasing amounts of mucopolysaccharide to a fixed amount of lectin. This interaction results in the development of a precipitin-like curve and leads to the isolation of a heparin fraction which has been found to be more reactive with respect to formation of a precipitate than the original heparin preparation. Monosaccharides such as α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine which are known to bind specifically to the lectin, greatly inhibit precipitate formation. The interactions between sulfated mucopolysaccharides and lectins have been used to isolate various sulfated mucopolysaccharides.     

Listeria species in a California coast estuarine environment.

Listeria species and L. monocytogenes were found in 81 and 62%, respectively, of fresh or low-salinity waters (37 samples) in tributaries draining into Humboldt-Arcata Bay, Calif., during a winter (January-February) sampling period. The incidence of Listeria species and L. monocytogenes in sediment (46 samples) from the same sites where water was sampled was 30.4 and 17.4%, respectively. One of three bay water samples contained Listeria species (including L. monocytogenes), while of 35 samples of oysters examined, only 1 was found positive for Listeria species (L. innocua). A given species or L. monocytogenes serogroup appeared to predominate in fresh water when domesticated animals (cows, horses) were nearby, whereas greater variety with no species predominance was observed in areas with no direct animal influence.     

Radioimmunoassay for prednisone

A radioimmunoassay for the measurement of prednisone in serum has been developed. The method is accurate, precise, specific, and very sensitive. Using a primary antibody elicited against prednisone 21-hemisuccinate-bovine serum albumin and a chemical precipitation step, the assay is capable of detecting 6.25 picograms of prednisone in 0.1 ml of unextracted diluted serum or plasma.The specificity of the assay is influenced by the carbonyl groups at position 11 and 20, and the double bond at the 1-position of the steroid nucleus. Physiological levels of endogenous steroid interfered only slightly with the primary antibody.Measurement of serum concentrations of prednisone in man and dog were accomplished following the administration of prednisone (Deltasone^®). This assay, used in conjunction with a published radioimmunoassay for prednisolone (1), can be used to determine the interconversion of these two drug products following prednisone or prednisolone administration.     

Majority of RNA which co-purifies with unactivated rat hepatic glucocorticoid-receptor complexes is nonspecific and not receptor-associated.

Molybdate-stabilized, unactivated rat hepatic glucocorticoid-receptor complexes were purified by a three-step procedure which includes affinity chromatography, gel filtration and anion exchange chromatography. Following elution of unactivated steroid-receptor complexes from the final DEAE-cellulose column, RNA which remained bound to the anion exchange resin was eluted with 1 M KCl. This RNA was small and heterogeneous in size. Equivalent amounts of RNA were detected after a mock purification which was devoid of receptors, suggesting that the presence of this RNA is not dependent on that of receptors. Both a [32P]DNA complementary to the RNA eluted from DEAE-cellulose and a [32P]DNA probe synthesized from total rat liver RNA gave similar results when hybridized to total rat liver RNA. These data indicated that the RNA which co-purified with unactivated receptors through the first two steps was very similar to total RNA in overall composition. Virtually identical hybridization patterns were also detected when end-labeled probes generated from the DEAE-cellulose eluted RNA or total liver RNA were hybridized to total genomic rat DNA, suggesting that the RNA eluted from the anion exchange resin is not specific or unique. Although these results do not exclude the possibility that there could be specific RNA species associated with the unactivated glucocorticoid receptor, they do indicate that the majority of the RNA eluted from DEAE-cellulose following elution of receptor complexes appears indistinguishable from total rat liver RNA and can be detected in parallel mock purifications.     

A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A

An alpha-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4(E249K), Pdcd4(D253A), Pdcd4(D414K), Pdcd4(D418A), Pdcd4(E249K,D414K), and Pdcd4(D253A,D418A)) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4(D253A) and Pdcd4(D253A,D418A) do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4(D253A), Pdcd4(D418A), or Pdcd4(D235A,D418A). Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.