Specific interaction of concanavalin a with glycolipid monolayers

The effect of ¹³¹I-labelled concanavalin A on the surface pressure and surface radioactivity of monolayers formed from phospholipids and from natural and synthetic glycolipids has been studied. The lectin binds to and penetrates dipalmitoyl phosphatidylcholine monolayers at a surface pressure of 15 dynes/cm and this interaction is inhibited by the presence of α-methyl mannose int he subphase. At surface pressures of 25 dynes/cm or higher, concanavalin A will interact with monoglucosyl diglyceride or diglucosyl diglyceride from Acholeplasma laidlawii and with synthetic glycolipids containing $\text{2}\text{or}\text{3 α1 → 4-}\text{linked}$ D-glucose residues in the headgroup, but not with phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or with the ganglioside II³NeuAc-GgOse₄-Cer. The binding to the glycolipid sugar group and penetration of the hydrocarbon region seem to occur simultaneously, as the time courses for the development of surface pressure and surface radioactivity coincide.     

Evidence for a convalent intermediate between alpha-glucosidase and glucose

A stable enzyme-glucose intermediate has been obtained in the short-term reaction between α-methyl-

-glucosidase and α-methyl-

-¹⁴C-glucopyranoside. A rapid-flow technique was employed in which phenol was used to terminate the reaction and to trap the product. It is believed that a covalent linkage is involved because (a) continued washing of the denatured protein failed to remove the radioactivity and (b) the radioactivity was retained by a tryptic peptide isolated by gel filtration. Treatment of the labeled protein with 2 $\text{N}$ HCl at room temperature released over 80% of the radioactivity as a compound with the same chromatographic mobility as glucose. No radioactive product was formed when bovine serum albumin replaced the enzyme, nor when glucosylamine, a potent glucosidase inhibitor, was present with the enzyme.     

Na+-dependent amino acid transport in preimplantation mouse embryos. II. Metabolic inhibitors and nature of the cation requirement.

Experiments were conducted in order to determine the energy source and nature of the cation dependency of [³H]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and ³H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport.The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na⁺-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K⁺-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K⁺-free medium in 1 hr. These results suggest that 1) Na⁺ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na⁺ pumps may be localized on the blastocoelic surface of the blastocysts.     

Immunologic suppression after oral administration of antigen. III. Activation of suppressor-inducer cells in the Peyer's patches

Mice were orally administered sheep erythrocytes (SRBC) in a regimen previously known to produce systemic tolerance to SRBC. Cellular interactions and movement from the gut-associated lymphoid tissue (GALT) to the spleen were found to occur using both in vivo and in vitro transfer systems. The cell in the GALT which initiates the suppression circuit migrates from the GALT to the spleen shortly after contacting antigen. This cell is a T suppressor-inducer (Tsi) cell which interacts with splenic lymphocytes to induce the formation of an effector T suppressor cell (Ts). The Tsi and Ts can be separated from each other by their differential sensitivities to cyclophosphamide. In addition, the Tsi can be separated from other GALT T cells by its inability to bind the lectin, peanut agglutinin. Thus, cell migration and cellular interaction among T cells must occur to result in orally induced tolerance.     

Sequence organization in Xenopus DNA studied by the electron microscope.

Xenopus laevis DNA was extracted from red blood cells and sheared to a mean length of 2780 nucleotides. The DNA was stripped of foldback-containing fragments and incubated to C₀t 10 (mol · s · l⁻¹), allowing most repetitive sequences to form duplex structures. Duplex-containing fragments were eluted from an hydroxylapatite column and visualized for electron microscopy by spreading from 57% formamide according to the modified Kleinschmidt technique of Davis et al. (1971). The mean length of the fragments observed was 2445 nucleotides. A total of 1700 DNA strands were photographed and studied. Less than 5% of the total strand length was in uninterpretable structures. Every molecule falling within the confines of the plates was included in the sample. Over 50% of the total strand length in the sample was found in structures bearing at least one interspersed repetitive sequence duplex terminated by four single-strand regions. The fraction of DNA present in duplex regions was almost exactly that predicted if the duplex regions represent all the interspersed middle repetitive sequence in the Xenopus genome. Direct measurement of visualized duplexes shows that the mean length of interspersed repetitive sequence elements in this genome is 345 nucleotides. Duplex length was shown to be independent of the length of the strands bearing the duplexes. These observations provide direct confirmation of the length of approximately 300 nucleotides indicated for interspersed repetitive sequences by earlier physical-chemical studies 011 Xenopus DNA. In strands carrying two duplexes terminated by single-strand regions the interduplex, or single-copy sequence element length could be measured. Sequence interspersion curves generated from these data are roughly consistent with those derived earlier from measurements of hydroxylapatite binding as a function of fragment length.     

The requirement for biotin and fatty acids in the cytotoxic T-cell response.

Dialyzed fetal calf serum (FCS) was a poor source of serum supplement for in vitro cytotoxic T lymphocyte (CTL) generation. Serum dialysate or biotin fully restored dialyzed FCS to activities comparable to FCS. It was concluded that the active principal in serum dialysate was biotin because its further dialysis was prevented by addition of avidin, a biotin binding protein. Avidin inhibited CTL generation only when added during the early stages of mixed lymphocyte cultures, whereas biotin could restore activity even if added at a later time. When FCS enriched in a fatty acid mixture, or in palmitic acid alone, was used as the serum supplement, avidin-mediated inhibition of CTL generation was markedly reduced. Avidin also inhibited CTL generation in cultures containing killed macrophages as the stimulating cell, and supplemented with Con-A-induced spleen cell supernatant, a source of helper factor(s). These experiments suggest that fatty acid biosynthesis and the attendant synthesis of structural lipids of appropriate fatty acid composition play a prominent role in the generation of CTL     

Chromosomal rearrangements which affect the chromosomal integration of the ribosomal genes in Drosophila melanogaster.

Sucrose gradient analysis of DNA from detergent-pronase lysates of whole adult flies has been used to examine a variety of genotypes for the presence of ribosomal genes not integrated into the DNA of the chromosome. Such genes were found in females in which one X chromosome carries an inversion, having one of its breakpoints between the nucleolus organizer and the centromere. These inversions move the nucleolus organizer to the distal end of the X chromosome. Other inversions which do not move the nucleolus organizer, as well as a series of bobbed deficiencies, did not induce unintegrated genes. The same inversions which induce unintegrated genes in adults also produce them in the diploid brain and imaginal discs of larvae. On the other hand, in the polytene salivary glands, unintegrated genes were found in every genotype examined.     

Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-k+-atpase-linked mechanism in blastocysts.

Mouse blastocysts collapse in cytochalasin B (CB), reexpand (accumulate fluid) in control medium, but cannot reexpand in ouabain, an inhibitor of Na⁺K⁺-ATPases. These ATPases, then, seem to be necessary for fluid accumulation in blastocysts. Since intact blastocysts are relatively insensitive to ouabain, CB seems to make it possible for ouabain to reach the Na⁺K⁺-ATPases localized on the blastocoelic surface. CB-Collapsed blastocysts were found to transport alanine and lysine at the same rate as intact blastocysts, indicating that, in 1 hr, amino acids are transported into the cells of the intact blastocyst, and not into the fluid-filled blastocoel. Transport rates in CB-collapsed blastocysts do not exceed those in intact blastocysts, suggesting that hypothetical amino acid carriers are located only on the external blastocyst surface. Most important, ouabain strongly inhibits sodium-dependent alanine transport in CB-collapsed blastocysts, but not in intact blastocysts, providing strong evidence that Na⁺K⁺-ATPases, localized on the blastocoelic surface, are necessary for this transport. Ouabain does not inhibit sodium-independent lysine transport in CB-collapsed blastocysts. Thus, the dependency of both sodium-dependent amino acid transport and fluid accumulation upon Na⁺K⁺-ATPases, and the separate localization of amino acid carriers and these ATPases, provides functional evidence for an epithelial tissue type of mechanism for sodium-dependent amino acid transport in mouse blastocysts.     

New methods for the purification of vertebrate vitellogenin.

Two procedures are described for the isolation of vitellogenin from plasma. Vitellogenin can be selectively precipitated from the plasma of estrogen-treated animals by Mg²⁺ in the presence of EDTA. The vitellogenin thus obtained is >99% pure and is relatively undegraded. In the second procedure, vitellogenin can also be isolated by DEAE-cellulose chromatography, resulting in preparations that contain <2% albumin. In combination, these two isolation procedures yield vitellogenin preparations of very high purity.     

Differences in the protein binding of several drugs and bilirubin in serum and heparinized plasma of rats.

The purpose of this investigation was to compare the protein binding of salicylic acid, phenytoin, warfarin and bilirubin in serum and heparinized plasma of rats. Protein binding was determined by equilibrium dialysis (drugs) or by a reaction rate method (bilirubin), using serum and plasma obtained from the same animals. The three drugs were significantly less protein bound in heparinized plasma than in serum; this difference was particularly pronounced in the case of warfarin. Addition of heparin to serum also resulted in a decrease in the protein binding of the drugs but to a lesser extent than in plasma. The protein binding of bilirubin was more extensive in plasma than in serum, irrespective of the anticoagulant used (heparin, sodium citrate, or disodium ethylenediamine-tetraacetate). It may be desirable to perform all binding studies with serum rather than plasma.     

Pyrimidine biosynthesis and its regulation in the estrogen-stimulated chick oviduct.

Studies on the incorporation of radio-labeled precursors into orotic acid and the pyrimidine nucleotides of RNA have established the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the chick oviduct. Measurements of the rate of incorporation of precursors into orotic acid in minces of oviduct revealed the activity of the orotate pathway to be accelerated in response to estrogen-stimulated nucleic acid synthesis and tissue growth. These data indicate that extrahepatic tissues of avian species meet their requirements for pyrimidine nucleotides through de novo synthesis rather than depend upon the liver or other exogenous sources for a supply of preformed pyrimidines. An examination of the influence of pyrimidine and purine nucleosides on the incorporation of radio-labeled precursors into orotic acid yielded evidence that pyrimidine biosynthesis in the chick is quite sensitive to inhibition by both purines and pyrimidines; the data indicate the reaction catalyzed by carbamoylphosphate synthetase to be the site of inhibition in both cases.     

Analogs of natural lipids. II. Polymorphic behavior of the tris-Homo acyl derivatives of cyclopentane-1,2,3-triols

The polymorphic behavior of the three series of tris-homoacyl (C_14:0, C_16:0 and C_18:0) cyclopentane-1,2,3-triol analogs of the natural saturated triglycerides has been studied using differential thermal analysis, Fourier transform infrared spectroscopy, and X-ray diffraction. It was found that the triglyceride analogs derived from the 1,2,3/0 and 1,2/3 cyclopentanetriols exhibit different polymorphic behavior than that of the natural triglycerides. The analogs derived from 1,3/2 cyclopentanetriol, however, were found to parallel the polymorphic behavior of the natural triglycerides quite closely. This polymorphic behavior is discussed in terms of the different configurations which the chains assume in each of the triglyceride analogs.     

T-lymphocyte/monocyte interaction in response to phytohemagglutinin.

Macrophage cooperation has been shown to be necessary for the thymus-derived lymphocyte to express many of its differentiated functions. The importance of macrophage-lymphocyte interaction has been extended to the study of nonimmunogenic mitogenesis. Utilizing human macrophages and T-cells prepared separately to greater than 98% purity, we have demonstrated a marked degree of dependence of the T-cell upon the macrophage for mitogenesis in response to phytohemagglutinin. The degree of dependence observed is greater than that seen with other human systems and on the order of that seen in the highly purified nonhuman systems. The nature of the physical interaction between the macrophage and the T-cell was visualized using conventional light microscopy and scanning electron microscopy. Lymphocytes were observed to form a semirosetting pattern around the macrophage very early following mitogenic stimulation. The lymphocytes were observed to proceed through early blastogenesis while in direct contact with the macrophage.