Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between <Emphasis Type="Italic">in situ</Emphasis> electroporation and retroviral transduction

Background  

Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.     

Prostaglandin generation in rabbit kidney. Hormone-activated selective lipolysis coupled to prostaglandin biosynthesis.

The endogenous release of prostaglandins and free fatty acids from the isolated perfused rabbit kidney in the absence or presence of stimulation by bradykinin or angiotensin-II was investigated. Basal (nonstimulated) release of prostaglandin-precursor arachidonic acid was 15-20-fold higher than that of prostaglandin E2 indicating a low conversion of released arachidonate to prostaglandins. Addition of bovine serum albumin to the perfusion medium caused a substantial (50-250%) increase in the release of all fatty acids except myristic and arachidonic acids, and no significant change in prostaglandin E2 generation. In contrast, administration of bradykinin (0.5 microgram) or angiotensin-II (1 microgram) caused a 10-15-fold increase in prostaglandin E2 release, and with albumin present, also a 2-3-fold selective increase in arachidonic acid release. Thus, unlike what was observed under basal conditions, arachidonic acid released following hormone stimulation is efficiently converted to prostaglandin E2. We conclude that administration of bradykinin or angiotensin-II into the perfused kidney activates a lipase which selectively releases arachidonic acid, probably from a unique lipid entity. This lipase reaction is tightly coupled to a prostaglandin generating system so that the released arachidonate is first made available to the prostaglandin cyclooxygenase, resulting in its substantial conversion to prostaglandins.     

Analysis by Single-Gene Reassortment Demonstrates that the 1918 Influenza Virus Is Functionally Compatible with a Low-Pathogenicity Avian Influenza Virus in Mice

The 1918-1919 "Spanish" influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight "7:1" chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.     

p38 MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells

We have previously reported that interleukin (IL)-1 beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1 beta (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 microM) decreased IL-1 beta-induced COX-2 by 70 +/- 7% (P < 0.01). SB-203580 had no effect on PGE(2) release in control cells but caused a significant (70-80%) reduction in PGE(2) release in IL-1 beta-treated cells. IL-1 beta increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-kappa B, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-kappa B activation. The NF-kappa B inhibitor MG-132 did not alter IL-1 beta-induced COX-2 expression, indicating that NF-kappa B activation is not required for IL-1 beta-induced COX-2 expression in HASM cells. IL-1 beta attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1 beta induces COX-2 expression, PGE(2) release, and beta-adrenergic hyporesponsiveness.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Rabbit esophagus metabolizes arachidonic acid predominantly via a lipoxygenase pathway

Eicosanoids modulate the response of gastrointestinal mucosa to noxious stimuli. Though these compounds have been extensively investigated in the stomach, their role in the esophagus has received less attention. Thus, the metabolism of 14C-arachidonic acid by homogenates of rabbit esophageal mucosa was investigated. The major metabolites formed and separated by TLC and HPLC had the chromatographic characteristics of (percent conversion follows each metabolite) 6-keto-prostaglandin F1 alpha (3.80 +/- 1.15), prostaglandin F2 alpha (2.05 +/- 0.37), prostaglandin E2 (5.92 +/- 1.65) and 12-hydroxyeicosatetraenoic acid (26.03 +/- 4.58). Indomethacin, a cyclooxygenase inhibitor, caused a significant decrease in prostaglandin formation without affecting 12-hydroxyeicosatetraenoic acid. BW755C, a combined cyclooxygenase-lipoxygenase inhibitor, dramatically decreased formation of all metabolites. It is concluded that esophageal mucosa metabolizes arachidonic acid primarily to a lipoxygenase derived product. This is the most abundantly produced eicosanoid yet described in the gastrointestinal tract. The importance of this compound to esophageal function is unknown but its presence suggests that future studies of eicosanoids in the esophagus should focus on lipoxygenase metabolites.     

Preparation and antitumor activity of nontoxic lipid A

Summary Highly refined, disaggregated endotoxic glycolipids (B5) from heptose-less (Re) mutant Salmonella typhimurium quantitatively converted to nontoxic (lethality for chick embryos) and nonpyrogenic (fever in rabbits) lipid A by treatment with boiling 0.1 N HCl (B5-HC1). Nontoxic B5-HCl, like toxic B5, caused regression of line-10 tumors and elimination of lymph node metastasis in 27 of 32 (84%) syngeneic strain 2 guinea pigs at a dosage of 150 g. At this dosage, toxic B5 led to a cure in 54 of 67 (81%) tumor-bearing animals. All cured animals rejected a second line-10 tumor cell transplant. This activity depended on combining the toxic or nontoxic endotoxins with mycobacterial trehalose mycolate (P3) and an essentially nontoxic peptide-containing side-fraction (ACP) recovered during the isolation of B5. In contrast to toxic B5 or endotoxins in general, nontoxic B5-HCl did not cause endotoxic shock when combined with adjuvant dipeptide (MDP) and injected IV into guinea pigs. Chemical analysis showed that the phosphate content of nontoxic B5-HCl was about one-half that observed in toxic B5 or in toxic KDO-free lipid A, which was obtained by treating toxic B5 with sodium acetate at pH 4.5 at 100° C (B5-pH 4.5). The molar ratio of glucosamine: phosphorus: fatty acids was 2:1:4 for nontoxic B5-HCl and was 2:2:4 for toxic B5-pH 4.5. These results demonstrate that endotoxic extracts could be selectively detoxified while retaining antitumor properties. Thus, nontoxic B5-HCl may be a potential candidate for immunotherapy of human cancer.Presented at the 72nd Annual Meeting of the American Association for Cancer Research, 1981, and abstract no. 1123 published in the Proceedings of the American Association for Cancer Research, Vol. 22, 1981 Abbreviations used in this paper: ACP, a nontoxic acetone-chloroform precipitated side-fraction of endotoxin that contains (an) ingredient(s) necessary for tumor regression of line-10 tumors in strain 2 guinea pigs; ReGl, endotoxic glycolipids from Re mutant gram-negative bacteria; ReGl-PCP, ReGl extracted with phenol-chloroform-petroleum ether (PCP); B5, refined endotoxin, free of phospholipids, divalent cations and disaggregated; B5-HCl, nontoxic lipid A prepared from B5 by treatment with hydrochloric acid; B5-pH 4.5, toxic lipid A prepared from B5 by treatment with sodium acetate at pH 4.5; lipid A, hydrochloric acid or sodium acetate hydrolysate of ReGl-PCP or B5; MDP, N-acetyl-muramyl-l-seryl-d-isoglutamine; KDO, keto-3-deoxyoctonate     

Neuromelanin: a role in MPTP-induced neurotoxicity

Methylphenyltetrahydropyridine (MPTP) selectively destroys melanin-containing neurons in the substantia nigra of humans and other primates. Methylphenylpyridine (MPP+), an active metabolite of MPTP, which is accumulated intraneuronally by the catecholamine uptake system, binds with high affinity to neuromelanin. MPP+ bound intracellularly to neuromelanin may be released gradually, resulting in damage to the neurons of the substantia nigra. Chloroquine, a drug which blocks MPP+ binding to neuromelanin, can protect monkeys from MPTP neurotoxicity.