Molecular heterogeneity of the concanavalin A tetramer: effects on binding to human red blood cells

We have found that the distribution of the three main monomer species found in tetrameric concanavalin A was approximately 73% type A monomer (27,000 MW); 4% type B monomer (14,000 MW); and 23% type C monomer (12,000 MW). When this tetrameric concanavalin A was bound to human erythrocytes and the monomer distribution of the bound concanavalin A was examined, we found that it resembled that of the concanavalin A used in the binding reaction. However, when competing sugars were used, either to inhibit the binding of concanavalin A or to remove previously-bound lectin, examination of cell-bound monomer distribution revealed that there was a significant increase in type C monomers and a simultaneous decrease in type A monomers. The shifts in monomer distribution varied depending on experimental conditions and the particular competing inhibitor employed. These findings were taken to indicate that not all concanavalin A cell surface interactions are identical and that quantitative methods are available for studying this phenomenon.     

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNA^Met_f has been further characterized. This substituent is responsible for the ¹⁹F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution ¹⁹F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield ¹⁹F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNA^Met_m and tRNA^Val_l, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with ¹⁹F spectrum of the model compound 5′-deoxy-5-fluoro-5,6-dihydrouridine (dH⁵₆FUrd) (δ_FUra = ? 31.4 ppm; J_HF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH⁵₆FUrd is considerably more alkali labile than 5,6-dihydrouridine (H⁵₆Urd). At pH 8.9, where H⁵₆Urd is stable, dH⁵₆FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a ¹⁹F resonance at ? 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The ¹⁹F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the ¹⁹F signal at ? 15 ppm. Hydrolysis of this tRNA with RNAase T₂ produces a sharp doublet 33 ppm upfield (J_HF = 45 Hz). Similarities of the ¹⁹F chemical shift and coupling constant to those of dH⁵₆FUrd, allows assignment of the peak at ? 33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.     

Triassic miospores from Southern Israel

Miospores collected from two boreholes that penetrated the Triassic sequence in southern Israel are systematically described, and their vertical distribution is shown. The assemblages comprise 71 morphospecies, of which 7 are proposed as new. These are: Alisporites brotzenii, Klausipollenites gerryi, Aculeisporites rosenbergii, Densosporites druckmanii, Uvaesporites sarahlei, Zonalapollenites zakii and Eucommiidites triassicus.     

Some comparisons between solution and crystal properties of thiosulfate sulfurtransferase

The activity and crystal stability of the enzyme thiosulfate sulfurtransferase were studied as a function of ionic strength. At 2 $\text{M}$ ammonium sulfate, where the x-ray structural studies of this protein were done soluble enzyme has low activity (<16% of the activity of the enzyme at an ionic strength of 0.1) and crystals of the enzyme are stable when substrates are added. However, at 1.4 M ammonium sulfate, crystals of TST rapidly dissolve in 1 mM CN^? but are relatively stable in 1 mM $\text{S}{}_2\text{O}{}_3{}^{\mathrm{=}}$. These results are consistent with a conformational change on converting the sulfur substituted form of the enzyme (ES) to the sulfur-free form (E) and helps to explain why this change was not observed in the crystallographic studies.     

Consumer (dis-)interest in genetic ancestry testing: the roles of race,immigration, and ancestral certainty

Genetic ancestry testing (GAT) is marketed as a way to make up for missing knowledge about one’s ancestry. Previous research questions the GAT industry’s ability to fulfill this promise in terms of the validity and reliability of test results. We instead explore the demand side of GAT, evaluating who is most and least likely to express interest in GAT. Using data from an original, nationwide survey of over 100,000 American adults, we find that GAT interest is related to both self-identified race and immigrant generation, with Asian Americans and first-generation immigrants expressing the least interest. Our quantitative and qualitative evidence suggests interest is further shaped by a pre-existing sense of ancestral certainty, leading some individuals to decline GAT, even if it were free. How interest and ancestral certainty are patterned has implications for who is included in – and thus for the conclusions that can be drawn from – genetic ancestry databases.     

Preformed GroES Oligomers Are Not Required as Functional Cochaperonins

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268–30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96–97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer–monomer equilibrium to be 7.3×10^–34M⁶. The GroESD(96–97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96–97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the preformed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.     

Ability of fixed B-lymphoma cells to present foreign protein antigen fragments and allogenic MHC molecules to a cloned helper-T-cell line

Cloned, L3T4+ T cells have been shown to respond to foreign protein antigens in the context of self-Ia glycoproteins and to non-self Ia glycoproteins. In the case of responses to foreign proteins, fixed antigen-presenting cells can present antigen fragments, but cannot present native proteins. Whether fixed allogenic cells can stimulate has been controversial. We have examined this question using a dual-reactive cloned helper-T-cell line. We find that conditions of fixation that block the presentation of native antigen to this cloned line, but which allow the presentation of antigen fragments, also allow presentation of allogeneic Ia molecules, leading to stimulation of the cloned line. This study also revealed an occult alloreactivity in the cloned T-cell line, which was expressed by fixed, but not by normal, antigen-presenting B lymphoma cells. All of these stimuli proceeded via the same clonotypic receptor, as determined by blocking with anti-T-cell receptor monoclonal antibody. These data suggest that responses to non-self Ia glycoproteins involve direct recognition of the allogeneic Ia molecules and do not require processing and presentation of these antigens by self Ia molecules.