T cell-activating protein on murine lymphocytes

A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression.     

Analysis of a large nontranscribed spacer in the ribosomal DNA of the house cricket,Acheta domesticus (Orthoptera:Gryllidae)

An analysis of a 29-kilobase nontranscribed spacer fragment in the ribosomal DNA (rDNA) of the house cricket, Acheta domesticus, revealed a highly repetitious structure. A total of eight EcoRI repeats of three different size classes measuring 259, 420, and 508 base pairs (bp) was mapped to a region 2 kilobases (kb) from the 18 S coding region. The repeats were oriented in a nonrandom manner and had sequences homologous to DNA located immediately adjacent to the repetitive array. DNA sequence analysis showed that the repetitive region was composed of smaller direct repeats 66, 67, and 383 bp in length. There was minor length heterogeneity of the chromosomal restriction fragments containing the entire array, indicating that a variable number of EcoRI repeats is a minor contributor to the total repeat-unit length heterogeneity. Immediately upstream from the EcoRI array there is a 17-kb region composed of 50 to 60 subrepeat elements recognized by a variety of restriction endonucleases. A subcloned SmaI repeat from the array was not homologous to any other part of the rDNA repeat unit or other chromosomal DNA. There was little length heterogeneity in restriction fragments containing the chromosomal 17-kb repetitions region. Immediately upstream from the 17-Kb region there is a 4.1-kb segment with sequences homologous to the EcoRI repeats.     

The expression, function, and ontogeny of a novel T cell-activating protein, TAP, in the thymus

The TAP molecule is an allelic 12,000 m.w. membrane protein that participates in T cell activation. This report analyzes the expression, function, and ontogeny of this molecule in the thymus. TAP is expressed on a small subset (10 to 20%) of thymocytes which is distinct from its expression on a majority (70%) of peripheral T lymphocytes. In the adult thymus, the majority of the TAP-bearing thymocytes are cortisone-resistant, Thy-1+, TL-, J11D-, and PNA-, which localizes TAP expression to medullary thymocytes. Cortical thymocytes do not bear this determinant. Parallel functional studies demonstrated that TAP+ thymocytes are required for Con A and MLR responsiveness. Anti-TAP MAb plus PMA specifically induces proliferation of mature thymocytes comparable in magnitude to the Con A response. These results demonstrate that TAP expression defines the immunocompetent thymocyte compartment and, further, that this molecule is functional on these cells. The ontogeny of TAP expression was also analyzed. TAP is expressed early in fetal thymic development at a time when most T cell markers (except Thy-1 and the iL2-R) are absent. The small sub-population of adult L3T4- and Lyt-2- thymocytes, which resemble early fetal thymocytes, also express TAP. These early thymocytes are capable of being activated through the TAP molecule. The implications of these findings for T cell development and, in particular, the relationship of TAP to T cell receptor expression and acquisition of immunocompetence are discussed.     

Studies on a Possible Functional Coupling Between Presynaptic Acetylcholinesterase and High-Affinity Choline Uptake in the Rat Brain

The relationships between presynaptic acetylcholinesterase (AChE) and high-affinity choline uptake (HACU) were investigated using a monolayer of rat cortex synaptosomes in superfusion conditions. The following sets of experiments were performed: determination of [3H]choline ([3H]Ch) uptake during superfusion with [3H]Ch; determination of [3H]Ch uptake during superfusion with acetylcholine (ACh) tritiated in the Ch moiety; evaluation of ACh hydrolysis during superfusion with ACh labelled in the acetate moiety; and comparison of the uptake of [3H]Ch generated by hydrolysis of [3H]ACh with that occurring during superfusion with [3H]Ch. Intact ACh was not taken up by superfused synaptosomes. The uptake of [3H]Ch during superfusion with 1 or 0.1 microM [N-methyl-3H]ACh was two-thirds of that occurring during superfusion with the same concentrations of [3H]Ch. The amount of [3H]Ch produced by hydrolysis during 16 min of superfusion was 1/25 of the amount passing through the synaptosomal monolayer during 16 min of superfusion with [3H]Ch. The results indicate that presynaptic AChE and HACU are located in close proximity to each other on the cholinergic terminal membrane, an observation suggesting the possibility of a functional coupling between the two mechanisms.     

Protein topography of the 40 S ribosomal subunit from Saccharomyces cerevisiae as shown by chemical cross-linking

Protein-protein cross-linking was used to examine the spatial arrangement of proteins within the 40 S ribosomal subunits of Saccharomyces cerevisiae. Purified ribosomal subunits were treated with either 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate under conditions such that the ribosomal particle was intact and that formation of 40 S subunit dimers was minimized. Proteins were extracted from the treated subunits and fractionated on Sephadex G-150 or by acid-urea-polyacrylamide gel electrophoresis. Cross-linked proteins in these fractions were analyzed by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Constituent members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Forty-two pairs involving 25 of the 32 40 S subunit proteins were identified. Many proteins were detected in several cross-linked dimers. These proteins with multiple cross-links form foci for the construction of a schematic model of the spatial arrangement of proteins within the 40 S subunit.     

A kinetic description of antifreeze glycoprotein activity

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.     

Interactions of permeant cations with sodium channels of squid axon membranes.

To determine how the permeant cations interact with the sodium channel, the instantaneous current-voltage (I-V) relationship, conductance-ion concentration relationship, and cation selectivity of sodium channels were studied with internally perfused, voltage clamped squid giant axons in the presence of different permeant cations in the external solution. In Na-containing media, the instantaneous I-V curve was almost linear between +60 and -20 mV, but deviated from the linearity in the direction to decrease the current at more negative potentials. The linearity of instantaneous I-V curve extended to more negative potentials with lowering the external Ca concentration. The I-V curve in Li solution was almost the same as that in Na solution. The linearity of the I-V curve improved in NH4 solution exhibiting only saturation at -100 mV with no sign of further decrease in current at more negative potentials. Guanidine and formamidine further linearized the instantaneous I-V curve. The conductance of the sodium channels as measured from the tail current saturated at high concentrations of permeant cations. The apparent dissociation constants determined from the conductance-ion concentration curve at -60 mV were as follows: Na, 378 mM; Li, 247 mM; NH4, 174 mM; guanidine, 111 mM; formamidine, 103 mM. The ratio of the test cation permeability to the sodium permeability as measured from the reversal potentials of tail currents varied with the test cation concentration and/or the membrane potential. These observations are incompatible with the independence principle, and can be explained on the basis of the Eyring's rate theory. It is suggested that the slope of the instantaneous I-V curve is determined by the relative affinity of permeant cations and blocking ions (Ca) for the binding site in the sodium channel. The ionic selectivity of the channel depends on the energy barrier profile of the channel.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Protein composition of the chromosomal scaffold and interphase nuclear matrix

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.     

Absolute configuration of the 5,6-oxide formed from 7,12-dimethylbenz[a]anthracene by cytochrome P450c

The absolute configurations of the enantiomeric 5,6-arene oxides of 7,12-dimethylbenz[a]anthracene (DMBA) were recently assigned such that the late eluting enantiomer from a chiral HPLC column has 5R,6S absolute configuration. [Mushtaq et al. (1984) BBRC 125, 539]. The authors further concluded that the 5R,6S-enantiomer predominates on metabolism of DMBA by cytochrome P450c in liver microsomes from 3-methylcholanthrene-treated rats. Their chemical assignment of absolute configuration is incorrect. Thus, metabolism of DMBA by these microsomes as well as by homogeneous cytochrome P450c produces 5,6-oxide highly enriched (95%) in the 5S,6R-enantiomer in accord with theoretical predictions.