Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Poly-N-Acetyllactosamine Glycans of Cellular Glycoproteins: Predominance of Linear Chains in Mouse Neuroblastoma and Rat Pheochromocytoma Cell Lines

To study the properties of protein-bound oligosaccharides in neuronally differentiating cells, two model systems were used: murine N1E-115 and N-18 neuroblastoma cells inducible by serum starvation and rat PC12 pheochromocytoma cells inducible by nerve growth factor. Glycopeptides were prepared from cells metabolically labeled with [3H]glucosamine and analyzed by gel filtration. The properties of the high-molecular-weight glycopeptides were studied using enzymatic digestion with neuraminidase and endo-beta-galactosidase. In contrast to other cell lines analyzed, the neuroblastoma and pheochromocytoma lines contained predominantly glycopeptides completely cleavable with endo-beta-galactosidase, which indicated that they were linear-type poly-N-acetyllactosamine glycans. The proportion of these linear chains in the high-molecular-weight fraction increased during neuronal differentiation in both cell systems. The linear nature of the glycans was also correlated with positive anti-i and negative anti-I reactivity of the cells in immunofluorescence microscopy. Specific cell surface labeling for poly-N-acetyllactosamine glycans and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several glycoprotein components, some of which showed changes during neuronal differentiation. The high proportion of linear poly-N-acetyllactosamine chains in these neuronal cell lines and its increase during neuronal differentiation suggests that these glycans may be a characteristic feature of neuronal or neuronally differentiating cells.     

Unsuccessful induction of low-zone tolerance to bovine serum albumin injected into the subarachnoid space

Introduction of T-dependent antigens into the subarachnoid space (isas) resulted in higher systemic antibody responses in mice than injections into the peritoneal cavity (ip) or other sites commonly used for immunization. Antibody production in isas immunized mice was not increased by treatment with cyclophosphamide (Cy) at doses known to abolish T-suppressor-cell activity, but such treatment increased antibody production in ip immunized mice toward the higher level which was observed in the isas immunized animals. Suppressor cell-dependent low zone tolerance (LZT) to TNP-BSA could not be induced by isas injections of deaggregated BSA (d-BSA). Conversely, mice which were unresponsive to ip injected d-BSA showed consistent systemic antibody responses when the antigen was injected isas. These observations indicate that immune responses initiated within the CNS are associated with relatively ineffective induction of systemic suppressor cell activity.     

Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

Differentiation antigens in human T-cell activation: Evidence that anti-VH antibodies react with a membrane structure on human T Lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series

The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human V_H antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin V_H fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIal, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIal and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.     

The Total Chemical Synthesis of Monocyte Chemotactic Protein-1 (MCP-1)

The affinity-based N^α-amino protecting group tetrabenzo [a,c,g,i]fluorenyl-17-methoxycarbonyl (Tbfmoc) has been utilized as a hydrophobic probe to allow the simple, quick and highly effective isolation of a 76 residue cysteine-containing protein (MCP-1). The base-labile Tbfmoc group can be removed under very mild conditions, which preserve the thiol-con taining protein in the reduced state. Oxidative folding was then used to furnish the biologically active β-chemokine MCP-1.     

Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.