Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.     

Cyclic AMP derivatives stimulate the chondrogenic differentiation of the mesoderm subjacent to the apical ectodermal ridge of the chick limb bud.

Recent studies indicate that one of the major functions of the apical ectodermal ridge (AER) of the embryonic chick limb bud is to maintain mesenchymal cells directly subjacent to it (i.e., cells extending 0.4-0.5 mm from the AER) in a labile, undifferentiated condition. Furthermore, when mesenchymal cells are freed from the AER's influence, either artifically or as a result of normal polarized proximal-to-distal limb outgrowth, they are freed to commence cytodifferentiation. In a preliminary attempt to investigate at a molecular level the mechanism by which the AER exerts its "negative" effect on the cytodifferentiation of subridge mesenchymal cells, we have examined the effect of a variety of agents that elevate cyclic AMP levels on the chondrogenic differentiation of the unspecialized subridge mesoderm of the limb bud in an organ culture system. Dibutyryl- and 8-hydroxy-cyclic AMP elicit a dose-dependent increase in the rate and amount of cartilage matrix formation and a corresponding dose-dependent increase in sulfated glycosaminoglycan accumulation by subridge mesoderm explants. The stimulatory effect of suboptimal concentrations of cyclic AMP derivatives is potentiated by the addition of theophylline. The stimulatory effect is limited to cyclic AMP derivatives, since dibutyryl-cyclic GMP and 5'-AMP have no effect. Thus agents that elevate intracellular cyclic AMP levels stimulate the chondrogenic differentiation of the unspecialized subridge mesoderm of the embryonic chick limb bud.     

Quantitative measurement of intracellular RNA genomes of Rauscher murine leukemia virus by competition hybridization in DNA excess.

The technique of competition hybridization in DNA excess was used to determine the intracellular distribution of RNA genomes of Rauscher murine leukemia virus. An examination of subcellular RNA fractions revealed that 59% of intracellular viral RNA genomes were associated with the nuclear-enriched fraction, 41% with the cytoplasmic fraction, and 18% with the polysomal-enriched fraction. Also, an analysis of total cellular RNA disclosed that 20% of intracellular viral RNA genomes were polyadenylated and 80% were not polyadenylated.     

Influence of temperature on ionic sparing effect and cell-associated cations in the moderate halophile, Micrococcus varians var. halophilus

Cells of the moderately halophilic Micrococcus varians var. halophilus grew well in a chemically defined medium containing 1 to 3 M NaCl and 0.0103 M K+. The requirement for NaCl could be partially replaced by K+,:Li+ and Cs+. The efficiency of the sparing effect of these cations for NaCl was in order of K+ GReater than Li+ greater than Cs+. Increase in growth temperature was found to enchance the sparing effect of Li+ and Cs+ but not that of K+. Over the range of NaCl concentrations in which the cells grew well, cell-Na+ concentrations were similar to the medium NaCl concentrations while cellK+ concentrations were several-fold that in the medium. Cell-bound Na+ and K+ concentrations increased proportionally with medium NaCl concentration and growth temperature. The temperature-dependent cation accumulation was more obvious with K+ than Na+. The cell-associated Na+ + K+ concentrations were almost as high as or slightly higher than the external media which contained appropriate levels of NaCl regardless of the growth temperature.     

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apoVLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellinin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.     

Mutator genes of baby hamster kidney cells.

Two mutator genes of mammalian cells were demonstrated. One was associated with the ribonucleoside diphosphate reductase, and the other was associated with an extreme adenosine sensitivity.     

Studies on the adrenal cortex of hypophysectomized rats: A model for abnormal cellular atrophy and death

Summary Biochemical and ultrastructural studies indicate that the atrophy of adrenal cortex in hypoyhysectomized rats involves the following changes: (1) One to two days after hypophysectomy, there is loss of template activity resulting from cumulative DNA-damage and heterochromatinization.In vivo ACTH-administration led to recuperation of these cells, indicating damage during hypophysectomized state to be reversible. (2) If the duration of hypophysectomy is prolonged, some of the cells become irreversibly damaged and can no longer recuperate afterin vivo ACTH administration. (3) The period of most rapid cell death is from the third to seventh day after hypophysectomy. The cause of cell death is probably due to membrane damage in the absence of protein synthesis, leading to lysis of the cells. Lysozomes and macrophages are apparently not involved.Supported by U.S.P.H.S. grants AM-5384 and AM-13724 and taken in part from dissertations submitted by Chan and by Mostafapour to Wayne State University in partial fulfillment towards the Ph.D. degree.An invited article.     

Specificity of two isolated wheat carboxypeptidases

The substrate specificity of two purified carboxypeptidases from germinated wheat has been examined. Both enzymes were active on a wide variety of carbobenzoxy substituted peptides but inactive with unsubstituted dipeptides. Neither enzyme was active upon endoprotease or amidase substrates and only low levels of esterase activity were evident. In time course studies, both enzymes gave rapid non-specific sequential release of amino acids, including proline, from the carboxyterminal of proteins and polypeptides of known amino acid sequence.     

Sesquiterpenes of thalloid liverworts of the genera Conocephalum,Lunularia, Metzgeria and Riccardia

Thalloid liverworts of orders Metzgeriales and Marchantiales elaborate essential oils distinguishable from those of the Jungermanniales by the absence of β-barbatene and anastreptene. Riccardia sinuata elaborates a novel tricyclic exomethylene sesquiterpene of as yet undetermined structure. Conocephalum conicum elaborates cadinene-type sesquiterpenes. β-Cadinene from the latter species is clearly enantiomeric to the same product from vascular plants.