Mitochondria and cell death.

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.

The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium anisopliae var acridum.

Topical application of Metarhizium anisopliae var acridum to the desert locust Schistocerca gregaria resulted in changes in the biochemistry and antimicrobial defenses of the haemolymph. M. anisopliae var acridum colonized the host haemolymph from day two post application. The haemocytes did not attach to, phagocytose or nodulate elements of the fungus. However, the presence of the fungus appeared to stimulate hemocyte aggregation over the first few days of mycosis though the number of aggregates declined subsequently. The total hemocyte count increased two days after application, indicating an overall stimulation of the immune system, but declined to a value below that for uninoculated controls by day four. The differential haemocyte count showed that the initial increase in total haemocyte count was primarily due to a larger number of coagulocytes. After day two consistent declines in cell number were observed for all haemocyte classes in mycosed insects. The activity of the enzyme, phenoloxidase, decreased during the course of infection. However, the converse was true for prophenoloxidase. Lysozyme levels were significantly smaller in infected than control locusts. There was a significant correlation between lysozyme and PO activities when data from mycosed and control insects were combined. The total protein content of the haemolymph decreased during the course of infection.

Biological mediators of insect immunity.

Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or hemocyte b1p4eins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. These responses are often accompanied by proteolytic activation of the phenoloxidase zymogen that is present in the hemolymph. A component of insect immune responses to bacteria is the synthesis by fat body and hemocytes of a variety of antibacterial proteins and peptides, which are secreted into the hemolymph. These molecules attack bacteria by several mechanisms. Inducible antifungal proteins have also been recently discovered in insect hemolymph. The promoters for several antibacterial protein genes in insects are regulated by transcription factors similar to those involved in mammalian acute phase responses.

The occurrence of inducible anti-Escherichia coli activity in hemolymph from the migratory grasshopper, Melanoplus sanguinipes.

1. Inducible antibacterial activity against an Escherichia coli strain has been shown to occur in the hemolymph of Melanoplus sanguinipes. 2. This anti-E. coli activity was induced by the infection with Serratia marcescens or E. coli K12 but not Enterobacter cloacae. 3. The greatest anti-E. coli activity was observed in hemolymph extracted from grasshoppers 6 hr after the injection of any of the two species of bacteria. 4. Anti-E. coli activity disappeared in hemolymph extracted from grasshoppers 8 hr after injection or longer.

Formation of an N-nitrosamine by oxidation.

Tolazamide, an antidiabetic drug, was found to produce N-nitrosohexamethyleneimine (NHM) upon exposure to an oxidizing agent and in the absence of a nitrosating agent. The oxidizing agents were either hydrogen peroxide or oxygen.

Does 5-hydroxytryptamine influence "purinergic" inhibitory neurons in the intestine?

Intrinsic inhibitory neurons to guinea pig taenia coli and small bowel circular muscle were activated by transmural electrical stimulation, and the postinhibitory contractile response of the muscle was utilized to evaluate whether or not the neuronal action of 5-hydroxytryptamine (5HT) was associated with the inhibitory neurons. The postinhibitory contractile responses of the small intestinal circular muscle were unaffected by 5HT. The 5HT antagonist methysergide also did not affect the poststimulus contractile response of the circular muscle. The amplitude and area under the contractile curve of the poststimulus contractile response of the taenia coli were reduced and the amplitude of the relaxation response to electrical stimulation was increased in one-half of the preparations after application of 5HT. Methysergide did not alter the poststimulus contractile response of the taenia coli. 5HT is implicated as a neurotransmitter substance for slow synaptic excitation within the enteric nervous system of the guinea pig small intestine; however, the 5HT synapses do not appear to be present on the "purinergic" inhibitory neurons nor on neurons that synaptically influence the inhibitory neurons.

Synthesis and biological activity of some antitumor benzophenanthridinium salts.

A facil synthesis of benzophenanthridinium salts has been developed and used for preparing a number of these compounds. The antitumor activities in mouse leukemia L1210 (LE) and P388 (PS) were determined as well as some selected antimicrobial activities. Although antitumor activity was exhibited by several of the derivatives, none was as active as the naturally occurring alkaloid fagaronine. Fagaronine was made available as a synthetic by an improved procedure. Some structure-activity relationships among antitumor benzophenanthridinium salts are discussed.