Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity

We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine [( 3H]TAZ) were treated with trypsin (T), alpha-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66,000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between pI 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P3 gamma revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms less than 47K and T-generated forms less than 31K) were no longer immunoreactive. In contrast, use of the antibody H222Sp gamma revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.     

Adaptation of the tetrazolium method for testing the seed viability, and scanning electron microscopy study of some Western European orchids

A modified tetrazolium method was formulated for use with seeds of Western European orchids. The sequence of treatments which gave the highest percentage of coloured (i.e. viable) embryos was: (1) pretreatment in a solution of 5% (w/v) Ca(OCl)₂+ 1% (v/v) Tween-80, (2) soaking for 1 day in sterile water, (3) the classical tetrazolium test. The optimal duration of the pretreatment in Ca(OCl)₂+ Tween-80 depends upon the species, and to investigate the effect a scanning electron microscopy study was performed on the testa of 3 species. For a given species, the optimal pretreatment period was not affected by the year of harvest or the source of the seed lots.     

Preservation of sorghum plant portions harvested,processed and ensiled at ten stages of maturity

Two varieties of grain sorghum were harvested at 10 intervals from 35–189 days post planting. Leaf, stem and head portions were separated before being prepared for chemical analysis or ensiled for 30 days in 1-1 silos with or without preservatives. The taller variety (FS-1b) accumulated 60% more dry matter than ORO-T with advancing plant maturity, while whole plant crude protein content decreased from near 20 to less than 7% for both varieties. Dry matter ensiling loss (DMEL) was different (P < 0.05) for each plant portion, but was lower and less variable after the 77-day harvest. Immature leaves and heads resulted in the greatest average DMEL of 31 and 24%, respectively. Propionic acid decreased DMEL, while an ammonia solution was ineffective when compared to control leaf, stem and heads. The DMEL of leaves was influenced (P < 0.05) by a varietal × modulus of fineness interaction while the stem exhibited an interaction with plant maturity × modulus of fineness. Modulus of fineness was not associated with levels of organic acid production in silages, but plant maturity significantly influenced acetic, propionic and butyric acid production in heads. These data indicated that numerous combinations of silage preservation techniques affected DMEL of sorghum plant portions at different maturities.     

Extracellular matrix metabolism by chondrocytes 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

THe incorporation of [³H]glycine into acid-insoluble protein and of [³H]acetate into glysoaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gardients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of poly-ribosomes that was reversed by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence on L-glutamine was also demonstrated for other avain connective tissues.     

Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.