Preparation and pharmacological evaluation of the R- and S-enantiomers of 3-(2-butylamino)-4H- and 3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide, two tissue selective ATP-sensitive potassium channel openers.

The preparation and the pharmacological evaluation of the R- and S-isomers of 3-(2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (BPDZ 42) and 3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (BPDZ 44), two potassium channel openers, is described. Their optical purity was estimated by means of capillary electrophoresis (R- and S-BPDZ 42) and chiral HPLC (R- and S-BPDZ 44). The absolute configuration of each isomer of BPDZ 44 was deduced from crystallographic data. Pharmacological assays performed with the R- and S-isomers of BPDZ 44 revealed only slight differences in their activity on pancreatic B-cells but significant differences in their activity on vascular smooth muscle cells: the R-isomer being sixfold more potent than its corresponding S-isomer. The R-isomer of BPDZ 42 was shown to be more potent than its corresponding S-isomer on the endocrine pancreas. S-BPDZ 44 as well as R- and S-BPDZ 42 were found to exhibit tissue selectivity for the pancreatic versus the vascular smooth muscle tissue.     

The remotor muscle of the lobster antenna: sarcoplasmic reticulum and skinned fiber experiments

The striated remotor muscle of the lobster antenna has an extraordinarily profuse sarcoplasmic reticulum as shown by electron microscopy. Gel electrophoresis reveals a simple protein composition in which the Ca2+-ATPase predominates. Vesicles of sarcoplasmic reticulum (SR) from this remotor are shown to operate Ca2+ binding, Ca2+ transport, and Ca2+-activated hydrolysis of ATP with an usual efficiency (2 Ca2+ transported per ATP hydrolysed, 4 mumol ATP hydrolysed/mg protein/min). Skinned fiber experiments were performed. They indicate behaviour of the remotor expected from observations by EM and gel electrophoresis: contraction of low maximal intensity under Ca2+ excitation, long internal diffusion time due to the large volume of SR to be crossed, and large Ca2+ content released in a caffeine-sensitive manner.     

Lipoxygenase- mediated cleavage of fatty acids in plant mitochondria

Incubation of cauliflower bud mitochondria in the presence of 5 mM CaCl2 results in a rapid hydrolysis of the main membrane phospholipsds. Under the action of phospholipase D, phosphatidic acid is produced and forms, within the membranes, a very labile complex with Ca2+ and HPO42-ions present in the incubation medium. With time, one observes a first step characterized by the formation of phosphatidic acid, followed by a second step linked to the breakdown of this phospholipid. The enzyme responsible for the disappearance of phosphalidic acid has been identified as lipoxygenase. In the presence of molecular oxygen, this enzyme acts on the polyun-saturated fatty acids of phosphatidic add (mainly C18:2 and C18:3) yielding small water-soluble molecules, one of them being identified as malondialdehyde (1, 3-propanedial). Experiments involving inhibitory conditions of the breakdown of phosphatidic acid indicate that lipoxygenase acts directly on membrane-bound phosphatidic acid without previous, involvement of a lipolytic acyl hydrolase activity. In addition, the lipoxygenase activity is fully sensitive to hydroxamate derivatives. It is proposed that the lipoxygenase activity may account for a part of the mitochondrial alternative electron pathway that is insensitive to cyanide.     

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

Immunologic effects of interleukin 2 in primary immunodeficiency diseases

Five children with primary deficiencies of T cell function were studied to assess the effects of highly purified exogenous Interleukin 2 (IL 2) on their in vitro T cell responses. The lymphocytes from one child with Nezelof's T cell deficiency demonstrated absence of endogenous IL 2 production and improved proliferative responses to mitogen or alloantigen in the presence of exogenous IL 2. Moreover, during in vitro mixed lymphocyte culture in the presence of exogenous IL 2, his lymphocytes were able to develop into cytotoxic effector cells. A second child with Nezelof's syndrome demonstrated a different type of defect. The lymphocytes from this child had less impairment of endogenous IL 2 production. Although IL 2 increased the proliferation of his cells in response to PHA, similar augmentation was not seen after stimulation with OKT3 or alloantigen. In cell-mediated cytotoxicity assays, after mixed lymphocyte culture, natural killer-like activity was strongly boosted in the cultures that contained IL 2, but T cell-mediated cytotoxicity was not. The lymphocytes from three patients with severe combined immunodeficiency did not show improved proliferative responses in the presence of IL 2. Thus, only one of the five patients demonstrated the combination of defective endogenous IL 2 production, but preservation of the ability to respond appropriately to exogenous IL 2. This child may therefore have suffered from a T cell defect pathophysiologically similar to that seen in nude or aged mice.     

Climate‐driven rampant speciation of the Cape flora

Aim To test whether the radiation of the extremely rich Cape flora is correlated with marine‐driven climate change. Location Middle to Late Miocene in the south‐east Atlantic and the Benguela Upwelling System (BUS) off the west coast of South Africa. Methods We studied the palynology of the thoroughly dated Middle to Late Miocene sediments of Ocean Drilling Program (ODP) Site 1085 retrieved from the Atlantic off the mouth of the Orange River. Both marine upwelling and terrestrial input are recorded at this site, which allows a direct correlation between changes in the terrestrial flora and the marine BUS in the south‐east Atlantic. Results Pollen types from plants of tropical affinity disappeared, and those from the Cape flora gradually increased, between 10 and 6 Ma. Our data corroborate the inferred dating of the diversification in Aizoaceae c. 8 Ma. Main conclusions Inferred vegetation changes for the Late Miocene south‐western African coast are the disappearance of Podocarpus‐dominated Afromontane forests, and a change in the vegetation of the coastal plain from tropical grassland and thicket to semi‐arid succulent vegetation. These changes are indicative of an increased summer drought, and are in step with the development of the southern BUS. They pre‐date the Pliocene uplift of the East African escarpment, suggesting that this did not play a role in stimulating vegetation change. Some Fynbos elements were present throughout the recorded period (from 11 Ma), suggesting that at least some elements of this vegetation were already in place during the onset of the BUS. This is consistent with a marine‐driven climate change in south‐western Africa triggering substantial radiation in the terrestrial flora, especially in the Aizoaceae.     

A rapid-filtration technique for membrane fragments or immobilized enzymes: measurements of substrate binding or ion fluxes with a few-millisecond time resolution

The construction and use of a filtration system with milliseconds time resolution is described here. This apparatus allows measurements of substrate binding to immobilized enzyme or ion fluxes through membrane vesicles to be performed over a very large time scale, from 10 ms to seconds. The main advantage of this system compared to the widely used quench-flow technique is that it does not require the use of an inhibitor. Following adsorption of the enzyme in an adequately chosen filter, the reaction is allowed to proceed within the filter during a forced filtration of a buffer containing the reactive substrate (or of a washing solution in the case of efflux measurements). The design allows the duration of filtration and buffer flux to be finely and reproducibly controlled. This paper illustrates the use of this rapid-filtration system for time-resolved measurements of calcium binding and transport by sarcoplasmic reticulum Ca-ATPase and of the initial phase of ADP transport by the ADP/ATP carrier of intact mitochondria.     

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation

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The first head-tailed virus,MFTV1, infecting hyperthermophilic methanogenic deep-sea archaea

Deep-sea hydrothermal vents are inhabited by complex communities of microbes and their viruses. Despite the importance of viruses in controlling the diversity, adaptation and evolution of their microbial hosts, to date, only eight bacterial and two archaeal viruses isolated from abyssal ecosystems have been described. Thus, our efforts focused on gaining new insights into viruses associated with deep-sea autotrophic archaea. Here, we provide the first evidence of an infection of hyperthermophilic methanogenic archaea by a head-tailed virus, Methanocaldococcus fervens tailed virus 1 (MFTV1). MFTV1 has an isometric head of 50 nm in diameter and a 150 nm-long non-contractile tail. Virions are released continuously without causing a sudden drop in host growth. MFTV1 infects Methanocaldococcus species and is the first hyperthermophilic head-tailed virus described thus far. The viral genome is a double-stranded linear DNA of 31 kb. Interestingly, our results suggest potential strategies adopted by the plasmid pMEFER01, carried by M. fervens, to spread horizontally in hyperthermophilic methanogens. The data presented here open a new window of understanding on how the abyssal mobilome interacts with hyperthermophilic marine archaea.