Indoxyl derivatives of drug metabolites

Indoxyl derivatives were detected as minor products among the urinary metabolites of two trial drugs, a benzodiazepine (GP 55 129) and a benzophenone (CGP 11 952). Their structures were elucidated by NMR and mass spectroscopy. Presumably, metabolites containing potential aldehyde functions react spontaneously with endogenous indoxyl. Such derivatives have not hitherto been encountered in drug metabolism.     

Technology and the family farm

It is one thing for innovation in agricultural practice to be implemented thoughtlessly; quite another for it to be opposed on principle. The introduction of bovine growth hormone is a case in point.     

Detection in vivo of protein-DNA interactions within the lac operon of Escherichia coli

Studies of the sequence-specific binding of proteins to DNA have so far relied on in vitro experiments using cloned restriction fragments containing the relevant DNA sequences. We have applied the genomic sequencing technique of Church and Gilbert to show that the interactions observed in vitro occur in vivo. We use this approach to study the binding of regulatory proteins to the lac operon in vivo and detect changes in the reactivity (inhibition or enhancement) of guanines to methylation by dimethyl sulphate caused by the proximity of proteins to the N-7 atom of these guanines. We can detect the simultaneous binding of the catobolite gene activator protein (CAP) and the Lac repressor to their specific recognition sequences, and following induction of the lac operon we observe effects that are related to RNA polymerase binding or RNA elongation. We have successfully used oligonucleotide probes as short as 17 bases to display genomic sequence.     

Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes

The lymphocyte function-associated molecule LFA-1 (CD11a/CD18) plays a key part in lymphocyte adhesion. Lymphocytes do not adhere spontaneously; activation of protein kinase C (PKC) by phorbol esters, however, gives rise to strong LFA-1-dependent adhesion, indicating that activation of LFA-1 is required to induce cell adhesion. We have now investigated whether the functionally important CD2 and CD3 surface structures on T lymphocytes are involved in the activation of LFA-1. The stimulation of these molecules, which causes activation of PKC, strongly promoted LFA-1-dependent adhesion. Furthermore, we demonstrate by using cells from an LFA-1-deficient patient that this enhanced lymphocyte adhesion is caused by activation of the LFA-1 molecule and not by activation of its ligands. LFA-1 was persistently activated by triggering through CD2 but only transiently by triggering through CD3. We postulate that CD2 and CD3 can differentially regulate the affinity of LFA-1 for its ligands by modulating its molecular conformation through PKC-dependent mechanisms.     

Mitogens trigger a calcium-independent signal for proliferation in phorbol-ester-treated lymphocytes

The activation of T lymphocytes by mitogens requires at least two signals; the first, delivered to T cells by a mitogen in conjunction with accessory cells (monocytes/macrophages), leads to the generation of the second signal, interleukin-2 (IL-2). The first signal also induces the expression of IL-2 receptors on the surface of a subpopulation of T cells; binding of IL-2 to its receptor then initiates a cascade of events culminating in DNA synthesis by these cells. Certain compounds act synergistically with mitogens in promoting T-cell proliferation by substituting for the activities of interacting cells or their products. For example, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been shown to restore the ability of macrophage-depleted T-cell populations to respond to mitogenic lectins. Transmembrane fluxes of calcium, leading to increased free cytosolic calcium concentrations ([Ca2+]), have been demonstrated following mitogen binding to lymphocytes and have been implicated in the initiation of cell proliferation. We show here that the effect of TPA on lymphocyte proliferation occurs in the absence of extracellular Ca2+ or detectable changes in [Ca2+]i, but only in the presence of mitogens. This suggests that in cells which have been incubated with the phorbol ester, mitogens can induce proliferation by a calcium-independent signal.     

Sodium deoxycholate promotes the absorption of heparin administered orally, probably by acting on gastrointestinal mucosa, in rats

Sodium deoxycholate (DOC), selected as a promoter of gastrointestinal absorption of heparin, was administered orally to rats, followed, at increasing intervals, by heparin. Maximal plasma clearing activity (PC) was obtained with a 60-min interval, though PC was still elicited after 24 h, suggesting that DOC acts on the gastrointestinal mucosa. Inhibition of blood coagulation was also observed after oral heparin. The suggestion that DOC increases heparin absorption is supported by increased plasma levels of heparin. No signs of several gastrointestinal damage were seen.     

A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.     

In vitro reactivation of anaphase spindle elongation using isolated diatom spindles

A key step for analysing the mechanochemistry of mitosis would be the isolation of a functional spindle capable of anaphase chromosome movement in vitro. Although Mazia and Dan first isolated spindles in 1952, with one or two possible exceptions, isolated spindles are non-functional. An alternative approach has used permeabilized cells to study anaphase chromosome movement, but these preparations are biochemically and morphologically complex, and hence difficult to analyse. We describe here a simple procedure for isolating diatom spindles which are capable of anaphase spindle elongation in vitro. With addition of ATP, the two half-spindles slide completely apart, with concomitant decrease in the zone of overlap. Electron microscopy reveals decreased numbers of microtubules throughout the spindle after ATP addition and confirms the complete absence of structures beyond the spindle poles. These results are inconsistent with theoretical models of mitosis which suggest that spindle poles are pushed apart by microtubule growth, are pulled apart by external forces applied to the poles, or are released from tension generated during spindle formation. The results are consitent with models that postulate mechanical interactions in the zone of microtubule overlap as a factor in spindle elongation.