Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label

A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed.     

Direct evidence for the existence of nominal antigen binding sites on T cell surface Ti alpha-beta heterodimers of MHC-restricted T cell clones

The binding of nominal antigen to Ti alpha-beta heterodimers on MHC-restricted human T cell clones specific for fluorescein-5-isothiocyanate (FL) was detected by flow cytometry and affinity chromatography. The FL-Ti interaction is of physiologic significance, since T cell activation is induced by cross-linked arrays of FL in the absence of the specific MHC recognition. High antigen valence is required to achieve stable binding to cells and subsequent activation, which is consistent with estimated Ti-FL association constants of less than 3 X 10(5) l/mol. In addition to providing direct evidence that the Ti alpha-beta heterodimer is the receptor for antigen, these data suggest that nominal antigen binding sites exist on the Ti molecules of at least some MHC-restricted clones.     

Column cellulose hydrolysis reactor: Cellulase adsorption profile

Summary A column cellulose hydrolysis reactor was set up using a single passage of cellulase enzyme which was followed with a continuous percolation of buffer. Hydrolysis rates were found to decline precipitously upon the removal of the non-adsorbed cellulase components. By comparing specific activities of the cellulase before and after adsorption on the cellulose column, it was concluded that the adsorption efficiencies for the cellulase components decreased from exoglucanase (1,4--d-glucan cellobiohydrolase EC 3.2.1.91) to endoglucanase [1,4-(1,3;1,4)--d-glucan 4-glucanohydrolase, EC 3.2.1.4] to -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.21). Of the adsorbed cellulase components, the rate of endoglucanase leaching from the cellulose column was 20 times that for the exoglucanase despite the greater adsorption efficiency of the latter. By analysing the cellulase components which were bound and not bound by the cellulose column and comparing them with a purified exoglucanase enzyme on sodium dodecyl sulfate polyacrylamide gels, it was confirmed that the major cellulase component adsorbed to the cellulose column was an exoglucanase component. The resultant loss of other cellulase components from the reactor was probably the cause for the much reduced rate of cellulose hydrolysis when these components were flushed out of the column.     

Column cellulose hydrolysis reactor: An efficient cellulose hydrolysis reactor with continuous cellulase recycling

Summary The use of a column cellulose hydrolysis reactor with continuous enzyme recycling was demonstrated by incorporating a continuous ultrafiltration apparatus at the effluent end of the column reactor. Using this setup, over 90% (w/v) cellulose hydrolysis was achieved, resulting in an average sugar concentration of 6.8% (w/v) in the effluent stream. The output of the system was 1.98 g of reducing sugar/l/h with a ratio of 87% (w/v) of the reducing sugars being monomeric sugars. Batch hydrolysis reactors were less effective, resulting in 57% (w/v) of the cellulose being hydrolyzed. The output of the batch reactor was 1.33 g of reducing sugar/l/h with similar product concentrations and percentage of monomeric sugars. The ratio of reducing sugar/filter paper unit of cellulase activity for the column method was 69.1 mg/U as compared to only 21.2 mg/U for the batch reactor.     

The significance of the aprotic solvent in monomer studies related to the primary subunit environment in the macromolecule

An advantage of aprotic polar solvent systems in the study of monomer interactions relevant to the macromolecular state is demonstrated with the measurement of nucleoside amino proton exchange rates in DMSO/water mixtures. The DMSO/water solvent provides the first unequivocal observation of general acid catalysis of nucleic acid amino proton exchange, which is undetectable in aqueous solution due to the formation of the endocyclic protonated nucleobase. Suppression of nucleobase protonation in the presence of buffer acid is a consequence of anion desolvation in the aprotic solvent. The detected route of general acid catalysis is demonstrated as a consequence of Watson-Crick H-bonding, leading to the implication that amino chemistry is modulated in the helical state to decrease amino proton lifetime in the closed macromolecular context of conformational information obtained by hydrogen exchange methods. This useful property of the aprotic solvent can be extended to monomeric studies pertaining to specific local site interactions affecting the function and conformation of proteins and nucleic acids.     

A possible phagocytic role for folliculo-stellate cells of anterior pituitary following estrogen withdrawal from primed male rats

Summary Ultrastructural changes suggesting a phagocytic role for the nongranular folliculo-stellate cells of the anterior pituitary are investigated in estrogen-primed male rats after withdrawal of estrogen. Morphological changes in mammotropes following the removal of a subcutaneous estradiol-containing Silastic implant include the formation of intracellular lipid bodies. These lipid bodies appear to be associated with enhanced estrogen-dependent prolactin secretion in mammotropes. Seven and 24 h after estrogen withdrawal intracellular lipid within mammotropes seems to be released into the intercellular space. Seventy-two h after estrogen withdrawal, lipid droplets are almost entirely cleared from mammotropes while folliculo-stellate cells become packed with lipid globules. Folliculo-stellate cells also undergo dramatic hypertrophy 7 and 24 h after the removal of E2-containing implants. Extensive intercellular junctions including zonulae adhaerentes, desmosomes, and putative gap junctions are formed. Intercellular junctions delineate extravascular channels into which numerous microvilli project. Folliculo-stellate cells appear capable of accumulating many lipid droplets, presumably related to mammotrope metabolism. What appear to be large secondary lysosomes as well as the lipid droplets are observed within folliculostellate cells; lipid, therefore, may be degraded through a lysosomal pathway in folliculo-stellate cells.     

Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia

Summary Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.     

Paper replication method for isolation of radiation-sensitive mutants.

A filter paper replication system particularly useful for isolation of radiation-sensitive mutants of pigmented bacteria was devised. The fidelity of replication was high. Adhesion between a paper disk and a properly dried master plate provided adequate contact pressure. The replicas arising from this technique constitute a convenient apparatus for general application in isolation of clones sensitive to a discriminating treatment.     

Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex

Recessive lethal mutations in the T/t-complex of the mouse characteristically show defective genetic complementation, even when they affect very different stages of embryogenesis and are known to be nonallelic. To address the question of their genetic or functional relationship, we have applied the cis-trans test, using several well defined recombinant t-chromosomes that carry two or more lethal mutations, and others that are devoid of specific lethals. We show here that the defective complementation that occurs between different t-lethals is a specific result of the trans configuration; thus these genes, which may map as much as 15 cM apart, constitute a functional unit. Some speculations are presented to interpret this enigma in terms of DNA plasticity.     

Structural correlates of cellular tumorigenicity and anchorage independence in transformed fibroblasts

The ability on nonhematopoietic cells to proliferate in vitro without attachment to a solid surface (anchorage independence) is known to be highly correlated with their ability to form tumors in nude mice. Transformed cells in vitro frequently also show less organization of the intracellular actin-containing microfilament bundles and less cell-surface fibronectin compared to normal cells. We have examined whether the loss of the anchorage requirement for growth is related to either of these structural changes in the cellular cytoskeleton. Our approach was to select a series of subclones from a nontransformed Syrian hamster fibroblast line, NIL8, for the acquisition of either anchorage independence in vitro or tumorigenicity in nude mice. These subclones were then examined for concomitant changes in the cytoskeletal structures. We found that anchorage independence, decreased actin cable organization, and tumorigenicity in nude mice were coordinately induced in both the in vitro- and in vivo-selected subclones, whereas the loss of fibronectin was not consistently coinduced with these three markers. These results suggest that the transformation-associated decrease in actin cable organization in this type of cell enhances the ability to grow without anchorage in vitro and to form tumors in vivo.