Asymmetric labeling of amino lipids in liposomes.

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H+ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin-K+ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents. When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching. These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surface, monitor vesicular fusion, and assess liposomal structure.     

Food selection by beavers

Summary Multidimensional contingency tables provide a powerful new statistical tool for analyzing interactions among several variables in an ecological study. This technique is applied to a study of selectivity in tree-cutting by two colonies of beavers in central Massachusetts.At the Blue Heron Cove colony, selection depends jointly on genus and diamter. Beavers cut birch of all diameters available but avoid large diameter maples, pines, and oaks. These beavers are choosy generalists: they show clear preferences in cutting various genera of trees for food yet they cut substantial numbers of trees of non-preferred genera. At the Tamplin Road Pond colony, discrete sites of concentrated cutting activity differ in genera and diameters of trees selected. Trees of all diameters are cut at one site close to water, small diameter trees are selected at two other sites farther from water. Ironwood is preferred at one site but selected against at two other sites. This difference between sites in generic selectivity has two plausible explanations: (1) tree species differ in nutritional and other chemical value between sites, (2) beavers sample trees of the various species present at some sites in order to assess the value of such sites as foraging areas.     

Surface structures and sense organs of the cypris larva of Balanus balanoides as seen by scanning and transmission electron microscopy.

Various features of the settlement stage larva (cyprid) of the barnacle, Balanus balanoides (L.), were studied using scanning and transmission electron microscopy. The cuticle of the valves is pitted and in section has a characteristic ultrastructure. Small sensory setae protrode from the surface of this cuticle and are probably mechanoreceptors able to sense water movement around the larva. Each of the pair of caudla appendages which protrude from between the larval valves posteriorly, is made up of several sensory setae. These appendages are able to sense settlement surface topography. Certain other features of the larva are alos described and their roles discussed; such features include the frontal filaments, antennules and thoracic limbs.     

Control of the antibody response of C57BL/6 Lyt-congenic strains to the Lyt-3.1 alloantigen by a gene linked to theLyt-2 locus

Congenic anti-Lyt-3.1 sera have recently been produced by immunizing B6-Lyt-2^a mice with thymocytes from either B6-Lyt-2^a, Lyt-3^a or B6-Lyt-2^a, Lyt-3^a, H-2^k mice (Boos et al. 1978). Surprisingly, mice of the congenic strain B6 failed to produce either anti-Lyt-2.1 or anti-Lyt-3.1 cytotoxic antibodies after identical immunizations. To determine the genetic basis for the difference in response to Lyt-3.1, (B6 × B6-Lyt-2^a)F_a mice and progeny of the backcross, (B6 × B6-Lyt-2^a)F₁ × B6-Lyt-2^a, were immunized with B6-Lyt-2^a, Lyt-3^a, H-2^k thymocytes. In addition, thymic biopsies of backcross progeny were performed and thymocytes tested for the Lyt-2.2 antigenic specificity. Results indicate that gene(s) governing the immune response to Lyt-3.1 is (are) linked to theLyt-2 locus, and that the responder allele (linked toLyt-2 ^a ) shows very poor penetrance in Lyt-2^a/Lyt-2^b mice.     

Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

A histofluorescence study of events accompanying accumulation and migration of norepinephrine within locally cooled nerves

Glyoxylic acid was used to induce fluorescence in sections of rabbit sciatic nerve. In fresh nerves treated with this agent there were scattered finely beaded axons with a weak blue-green fluorescence. During local cooling, blue—green fluorescence accumulated steadily at the proximal boundary of the cooled region but never at its distal boundary. This accumulation gave rise to dilated axons that often swelled into brilliantly fluorescent balloon-like structures up to 10 μm in diameter. Axonal fluorescence was probably specific for norepinephrine, being enhanced by inhibition of the metabolism and diminished by inhibition of the synthesis or storage of this neurotransmitter. After local cooling of nerves for 1.5 hr, specific fluorescence was confined within 0.8 mm of the cooled region. Rewarming led to rapid removal of fluorescence from the cooled region and to disappearance of most of the balloon-like swellings. Simultaneously, rewarming caused brightly fluorescent fibers that were neither dilated nor swollen to appear in distal regions of nerve. As this wave of fluorescence migrated distally with increasing duration of rewarming, it was spread over increasingly broad regions of nerve, which suggests that axonal transport of norepinephrine may involve some kind of dispersive process.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

Regulatory factors produced by lymphocytes. I. The occurrence of multiple alpha-lymphotoxins associated with ribonuclease activity.

Supernatant fluids of mitogen-activated human tonsil lymphocytes contain large amounts of a factor toxic to mouse L cells. This substance, with a m.w. of 80,000 +/- 5,000 daltons, is called alpha-lymphotoxin (alpha-LT), to differentiate it from another toxin elaborated by mitogen activated human blood lymphocytes, called beta-lymphotoxin (beta-LT), which differs from alpha-LT in size (45,000 +/- 5,000 daltons), antigenicity, and stability. Further purification of alpha-LT by sequential phosphocellulose and DEAE-cellulose chromatography and polyacrylamide gel electrophoresis (PAGE) identifies a series of cytotoxins differing in ion exchange characteristics and electrophoretic mobilities. The three PAGE fractions (PAGE Ia, Ib and II), recovered in 2, 4.6, and 21% yield from the starting serum-free culture supernatant, represent purifications of 24-, 24- and 1851-fold, respectively. Each cytotoxic fraction has a ribonuclease activity. Comparison of RNase and mouse L cell cytotoxic activities of the three alpha-LT fractions shows that both activities for all three fractions have a similar temperature stability pattern and that both are similarly inhibited by DNA, single strand forms better than double strands, by glycerol in 5 to 20% concentration, and by protein denaturing reagents. These observations suggest, but do not prove, that mouse L cell toxicity and RNase activity are mediated by the same substance, which appears to occur in multiple or isozymic forms.