The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

No requirements of cyclic conformation of antagonists in binding to vasopressin receptors

Early reports that acyclic analogues of oxytocin and vasopressin (AVP) have drastically reduced agonistic activities established as dogma that an intact hexapeptide ring structure is essential for the pharmacological activities of analogues of neurohypophysial hormones. Thus, virtually all the many hundreds of agonistic and antagonistic analogues of the neurohypophysial peptides that have been reported contain an intact ring. Here we report that an intact ring is not essential for binding of antagonistic AVP analogues to vasopressor (V1) or antidiuretic (V2) AVP receptors. In fact, one acyclic AVP analogue seems to be about as potent as any previously reported cyclic V2 antagonist. This finding suggests new possibilities for the design of AVP analogues as pharmacological probes and for therapeutic use. Similar modifications might be useful in the design of analogues of other cyclic peptides, such as calcitonin, somatostatin and the atrial natriuretic factors.     

Expression and functional characterization of artificial mutants of interleukin-2 receptor

The physiological proliferation of T lymphocytes (T cells) requires interaction between the humoral growth factor, interleukin 2 (IL-2) and its cell-surface receptor. Studies of IL-2 binding to the IL-2 receptor (IL-2R) on T cells have revealed that there are two distinct species of IL-2R, one with high and one with low affinity. Isolation and characterization of cDNA for the human IL-2R made it possible to deduce the complete primary sequence (251 residues) of the receptor protein. However, expression of IL-2R alone is not sufficient for either growth signal transduction or high-affinity site formation: another lymphocyte-specific molecule called converter seems to be required for the biological activity of IL-2R. We found that the converter did not form a stable complex with IL-2R unless the receptor bound the ligand (the 'affinity conversion' model). To discover which are the functionally important parts of the human IL-2R we have constructed artificial mutant cDNAs encoding the receptor. The mutant receptors produced from them had deletions or substitutions in the cytoplasmic region (13 residues), the transmembrane region (19 residues) or the carboxy-terminal portion of the extracellular region (219 residues). All were active in growth signal transduction, efficient internalization and high-affinity site formation in two mouse T-cell lines, suggesting that the extracellular region of IL-2R and the converter may be responsible for growth signal transduction.     

Identification of receptor contact site involved in receptor-G protein coupling

The mammalian G proteins transduce information from extracellular signals, including neurotransmitters, hormones and sensory stimuli, into regulation of effector enzymes or ion channels within cells. Triggered by appropriate extracellular signals, receptor proteins specifically activate members of the G protein family by catalysing replacement of GDP by GTP at the guanine nucleotide binding site. Like the receptor proteins, the heterotrimeric G proteins exhibit impressive structural similarities, suggesting that all receptor-G protein interactions use homologous structural elements and a single molecular mechanism. Topologically equivalent portions of each G protein may therefore interact with the appropriate receptor. We recently predicted the secondary structure of a composite G protein alpha-chain and proposed that a predicted amphipathic alpha-helix at the extreme carboxy-terminus of the polypeptide directly contacts receptors. This proposal has now been confirmed by sequencing complementary DNAs of the gene that encodes the alpha-chain (alpha s) of the stimulatory regulator (Gs) of adenylyl cyclase in wild-type cells and in a mutant mouse S49 lymphoma cell line, unc, in which Gs cannot be activated by hormone receptors. The sequences reveal a point mutation in the unc gene that substitutes a proline residue for an arginine near the carboxy-terminus of the alpha s-polypeptide. Expression of recombinant alpha s-unc in genetically alpha s-deficient S49 cells reproduces the unc phenotype.     

A new immunomodulating compound (AS-101) with potential therapeutic application

There has been interest in the potential of synthetic compounds to modify immune responses by imitation of cytokine action. Direct administration of interleukin 2 (IL-2) in conjunction with adoptive transfer of lymphokine activated killer cells has been used in the treatment of cancer, but there are toxic effects resulting from the high doses of IL-2 required. We have developed a new synthetic compound, ammonium tri-chloro(dioxoethylene-O,O'-)tellurate (AS-101), which has immunomodulating properties and minimal toxicity. The effects of AS-101 on the activation and function of immunocompetent cells have been assessed. We have found that AS-101 induces proliferation and IL-2 production by human lymphocytes in vitro, and enhances the production of IL-2 and colony-stimulating factor by mouse spleen cells. Splenocytes of BALB/c mice injected with AS-101 increased production of IL-2 and CSF in vitro in the presence of mitogen. Mononuclear cells of normal donors acquired responsiveness to recombinant IL-2 and bound monoclonal antibody to IL-2 receptor after incubation with AS-101. Splenocytes of mice treated in vivo with AS-101 expressed high levels of IL-2 receptor. The stimulation of lymphocytes by AS-101 apparently involves an increase in intracellular free calcium. AS-101 administered systemically to mice mediated antitumour effects which could be attributable to its immunomodulatory properties. In addition, AS-101 could directly enhance the ratio of OKT4 to OKT8-positive cells in cultured mononuclear cells from AIDS (acquired immune deficiency syndrome) patients. These results indicate that AS-101 is potentially useful in the treatment of clinical conditions involving immunosuppression.     

Micromolar concentrations of Zn2+ antagonize NMDA and GABA responses of hippocampal neurons

NMDA (N-methyl-D-aspartate) receptors serve as modulators of synaptic transmission in the mammalian central nervous system (CNS) with both short-term and long-lasting effects. Divalent cations are pivotal in determining this behaviour in that Mg2+ blocks the ion channel in a voltage-dependent manner, and Ca2+ permeates NMDA channels. Zn2+ could also modulate neuronal excitability because it is present at high concentrations in brain, especially the synaptic vesicles of mossy fibers in the hippocampus and is released with neuronal activity. Both proconvulsant and depressant actions of Zn2+ have been reported. We have found that zinc is a potent non-competitive antagonist of NMDA responses on cultured hippocampal neurons. Unlike Mg2+, the effect of Zn2+ is not voltage-sensitive between -40 and +60 mV, suggesting that Zn2+ and Mg2+ act at distinct sites. In addition, we have found that Zn2+ antagonizes responses to the inhibitory transmitter GABA (gamma-aminobutyric acid). Our results provide evidence for an additional metal-binding site on the NMDA receptor channel, and suggest that Zn2+ may regulate both excitatory and inhibitory synaptic transmission in the hippocampus.     

Millisecond X-ray diffraction and the first electron density map from Laue photographs of a protein crystal

Attempts to use X-ray crystallography to extract three-dimensional information on transient phenomena in crystals have been hampered primarily by long data collection times. Here we report on the first difference Fourier map obtained from Laue diffraction photographs of a protein crystal, glycogen phosphorylase b. Data collection time was 3 s using the high-intensity white X-radiation generated on the wiggler magnet of the Daresbury Synchrotron Radiation Source (SRS), but data acquisition in the millisecond-submillisecond range is possible. The method presented here uses a simple difference technique and was designed to analyse structural changes relative to a known starting structure. The combination of this approach with cine techniques allows the recording of three-dimensional motion pictures at atomic resolution and opens up new areas in structural biology and chemistry.     

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.     

The role of clonal selection and somatic mutation in autoimmunity

Polyclonal activation has been proposed as the reason that autoantibodies are produced during autoimmune disease. This model denies a role for specific antigen selection of B cells and predicts instead a multiclonal population of unmutated or randomly mutated autoantibodies. We have found that the genetic features and clonal composition of spontaneously derived immunoglobulin G (IgG) antiself-IgG (rheumatoid factor (RF] autoantibodies derived from the autoimmune MRL/lpr mouse strain are inconsistent with both the predictions of this model and the actual outcome of experimental polyclonal activation. Instead we have found that MRL/lpr RFs are oligoclonal or even monoclonal in origin. They harbour numerous somatic mutations which are distributed in a way that suggests immunoglobulin-receptor-dependent selection of these mutations. In this sense, the MRL/lpr RFs resemble antibodies elicited by exogenous antigens after secondary immunization. The parallels suggest that, like secondary immune responses, antigen stimulation is important in the generation of MRL/lpr RFs.