Glucocorticoid-induced lymphocytolysis: State of the genetic analysis

The glucocorticoid-induced lysis of lymphoid cell lines offers a genetic approach to steroid hormone action because unresponsive variants can easily be selected as resistant to this lytic effect. The present state of analysis of lymphocytolysis in two murine cell lines, the S49 T-lymphoma and the W7 thymoma, is reviewed. All glucocorticoid-resistant variants isolated so far result from various defects in the glucocorticoid receptor. The absence of variants blocked at another step of the lytic mechanism is discussed. The observed hemizygosity of the glucocorticoid receptor locus in the S49 line and the instability of cell hybrids illustrate some of the potential problems encountered in somatic cell genetics.     

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.     

Contraction of bovine coronary vascular smooth muscle induced by cocaine is not mediated by norepinephrine

This study was designed to assess the effects of cocaine on coronary arterial smooth muscle and to determine whether previously reported cocaine-induced coronary vasospasm is mediated by substances released from the endothelium or by increased adrenergic receptor stimulation. Concentration-response relationships for cocaine (0.1-300 microM) and norepinephrine (0.1-300 microM) were studied in vitro using 2 mm segments of bovine proximal left anterior descending coronary artery. Each segmental ring was mounted in a 70 ml tissue bath for the measurement of isometric tension. Cocaine (3-300 microM) caused significant, concentration-dependent, increases in developed tension (p less than 0.05). Removal of the endothelium or pretreatment with prazosin (0.1 microM) or propranolol (1 microM) did not significantly alter this action of cocaine. In contrast to cocaine, norepinephrine (10-300 microM) caused significant decreases in developed tension (p less than 0.01). These findings suggest that cocaine-induced contraction of bovine coronary vascular smooth muscle is not mediated by endothelium derived contracting substances or norepinephrine.     

Tolerance induced by chronic inhaled antigen in a murine asthma model is not mediated by endotoxin

Ovalbumin (OVA)-sensitized wildtype (WT) and endotoxin-resistant (ER) mice developed similar degrees of airways eosinophilia and serum OVA-specific IgE levels after acute aerosolized OVA challenge. WT mice demonstrated methacholine hyperreactivity, whereas ER mice showed no change in responsiveness. With chronic aerosolized OVA challenge, both WT and ER mice developed local tolerance, with resolution of airway eosinophilia but persistence of anti-OVA IgE in serum. Thus, the development of local tolerance with chronic aerosol exposure to OVA is independent of any potential effects of endotoxin in the OVA aerosol solution.     

Gastric cytoprotection by pirenzepine is not mediated by catecholamines

The effects of pirenzepine (in a dose of 25.0 mg X kg-1) and atropine (2.5 mg X kg-1) were studied on the development of gastric ulceration produced by pylorus ligation, polymyxin B and absolute ethanol, as well as on the gastric secretory responses and plasma level of noradrenaline. It was found that: (1) pirenzepine significantly decreased the development of ulcer formation produced by pylorus ligation, polymyxin B and absolute ethanol without any antisecretory response; (2) atropine inhibited gastric acid secretion, but no effect was obtained on ulcus produced by pylorus ligation, polymyxin B and absolute ethanol; (3) the plasma level of noradrenaline could be decreased by atropine and pirenzepine, although the difference did not reach statistical significance. It has been concluded that catecholamines are not involved in the gastric cytoprotective mechanism of pirenzepine.     

Site-specific endonuclease NspLKI is an isoschizomer of endonuclease HaeIII

Site-specific endonuclease NspLKI has been isolated and purified to functionally pure state from soil bacterium Nocardia species LK by successive chromatography on columns with phosphocellulose, HTP hydroxyapatite, and heparin-Sepharose. The isolated enzyme recognizes the 5'-GG downward arrowCC-3' sequence on DNA and cleaves it as indicated by the arrow, i.e., it is an isoschizomer of HaeIII. The final enzyme yield is 1.105 units per gram of wet biomass. The enzyme is active in the temperature range of 25-60 degrees C with an optimum at 48-55 degrees C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM Tris-HCl, pH 7.4, 200 microgram/ml albumin, 10 mM MgCl2, and 100 mM NaCl.     

Stress-induced apoptosis is not mediated by endolysosomal ceramide.

A major lipid-signaling pathway in mammalian cells implicates the generation of ceramide from the ubiquitous sphingolipid sphingomyelin (SM). Hydrolysis of SM by a sphingomyelinase present in acidic compartments has been reported to mediate, via the production of ceramide, the apoptotic cell death triggered by stress-inducing agents. In the present study, we investigated whether the ceramide formed within or accumulated in lysosomes indeed triggers apoptosis. A series of observations strongly suggests that ceramide involved in stress-induced apoptosis is not endolysosomal: 1) Although short-chain ceramides induced apoptosis, loading cells with natural ceramide through receptor-mediated endocytosis did not result in cell death. 2) Neither TNF-alpha nor anti-CD95 induced the degradation to ceramide of a natural SM that had been first introduced selectively into acidic compartments. 3) Stimulation of SV40-transformed fibroblasts by TNF-alpha or CD40 ligand resulted in apoptosis equally well in cells derived from control individuals and from patients affected with Farber disease, having a genetic defect of acid ceramidase activity leading to lysosomal accumulation of ceramide. Also, induction of apoptosis using anti-CD95 (Fas) or anti-CD40 antibodies, TNF-alpha, daunorubicin, and ionizing radiation was similar in control and Farber disease lymphoid cells. In all cases, apoptosis was preceded by a comparable increase of intracellular ceramide levels. 4) Retroviral-mediated gene transfer and overexpression of acid ceramidase in Farber fibroblasts, which led to complete metabolic correction of the ceramide catabolic defect, did not affect the cell response to TNF-alpha and CD40 ligand.     

Pial arteriole dilation during somatosensory stimulation is not mediated by an increase in CSF metabolites

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to contralateral sciatic nerve stimulation. The mechanism of this pial vasodilation is not well understood. One possibility is that vasoactive metabolites released during brain activation may diffuse to subarachnoid cerebrospinal fluid (CSF) to dilate pial vessels. To test this hypothesis, we implanted closed cranial windows in rats and measured pial arteriolar dilation to sciatic nerve stimulation during constant rate superfusion of the pial surface with artificial CSF. We reason that flushing the pial surface with CSF should quickly dissipate vasoactive substances and prevent these substances from dilating pial arterioles. CSF flow (1 and 1.5 ml/min) significantly reduced pial arteriole dilation induced by 5% CO2 inhalation, but the same flow rates did not affect dilator responses to sciatic nerve stimulation. We conclude that brain-to-CSF diffusion of vasoactive metabolites does not play a significant role in the dilation of pial arterioles during somatosensory activity.     

Lethal graft-vs-host disease induced by a class II MHC antigen only disparity is not mediated by cytotoxic T cells

Treatment of C57BL/6J (B6) murine splenocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes NK cells, CTL precursors, and the capacity to cause lethal graft-vs-host disease (GVHD) in irradiated B6 X DBA/2 F1 mice. In contrast, alloantigen-induced L3T4(+) Th cell function has been shown to be relatively preserved after exposure to this agent. The present studies assessed the effects of Leu-Leu-OMe treatment of donor cells on induction of lethal GVHD in other murine strain combinations. When irradiated B6 X CBAF1 mice were infused with T and NK cell-depleted B6 bone marrow cells and 3 to 30 X 10(6) B6 spleen cells, uniformly lethal GVHD was observed. However, B6 X CBAF1 recipients of T and NK-depleted B6 bone marrow cells and similar numbers of Leu-Leu-OMe-treated B6 spleen cells demonstrated 90 to 100% long term survival. In contrast, Leu-Leu-OMe treatment of B6 donor cells had no beneficial effect on mortality rates in irradiated (B6 X B6-C-H-2bm12)F1 (B6 X bm12F1) recipients. When B6 spleen cells were stimulated in vivo or in vitro with either B6 X CBAF1 or B6 X bm12F1 stimulator cells, the capacity to generate alloantigen-specific CTL was abolished comparably by Leu-Leu-OMe treatment. Thus, the dramatic difference between the effects of Leu-Leu-OMe treatment of B6 spleen cells on the course of GVHD in B6 x CBAF1 and class II MHC only disparate B6 x bm12F1 recipients could not be explained by unique resistance of bm12-specific CTL precursors to Leu-Leu-OMe. These findings indicate that T cell effector mechanisms distinct from classic cell-mediated cytotoxicity are sufficient to generate lethal GVHD in class II MHC only disparate B6----B6 X bm12F1 mice.     

Colicin E3 is an endonuclease.

It was confirmed by polyacrylamide gel electrophoresis that isolated 16S rRNA was cleaved by the active component (protein A) or the active fragment (T2A) of colicin E3. However, the degradation was random, in contrast with the specific cleavage observed in the interaction of colicin E3 with ribosomes. Furthermore, the active component and the active fragment had low activities, and far greater amounts of these materials were required for degradation of the isolated rRNA than for ribosome inactivation. The degradation of rRNA cannot be due to contaminating ribonuclease(s), but is due to colicin E3 itself, because of the following facts. (1) Protein B of colicin E3, which specifically inhibits the ribosome-inactivating activity of colicin E3, inhibited the degradation of rRNA. (2) Protein B of colicin E2, which inhibits the action of colicin E2 but not of colicin E3, failed to inhibit the degradation of rRNA. (3) The activity appeared in the peak of protein A or fragment T2A, respectively, when they were rechromatographed on Sephadex G-75.     

Plant green-island phenotype induced by leaf-miners is mediated by bacterial symbionts

The life cycles of many organisms are constrained by the seasonality of resources. This is particularly true for leaf-mining herbivorous insects that use deciduous leaves to fuel growth and reproduction even beyond leaf fall. Our results suggest that an intimate association with bacterial endosymbionts might be their way of coping with nutritional constraints to ensure successful development in an otherwise senescent environment. We show that the phytophagous leaf-mining moth Phyllonorycter blancardella (Lepidoptera) relies on bacterial endosymbionts, most likely Wolbachia, to manipulate the physiology of its host plant resulting in the ‘green-island’ phenotype—photosynthetically active green patches in otherwise senescent leaves—and to increase its fitness. Curing leaf-miners of their symbiotic partner resulted in the absence of green-island formation on leaves, increased compensatory larval feeding and higher insect mortality. Our results suggest that bacteria impact green-island induction through manipulation of cytokinin levels. This is the first time, to our knowledge, that insect bacterial endosymbionts have been associated with plant physiology.     

Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release

Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia or the murine N9 microglial cell line with chromogranin A resulted in nitric oxide production, which triggered microglial apoptosis. Exposure of microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity.     

Evidence that histamine-stimulated prolactin secretion is not mediated by an inhibition of tuberoinfundibular dopaminergic neurons.

The effects of histamine on prolactin secretion and the activity of tuberoinfundibular dopaminergic (DA) neurons were examined in male rats. Tuberoinfundibular DA neuronal activity was estimated in situ by measuring the metabolism [concentration of 3,4-dihydroxyphenylacetic acid (DOPAC)] and synthesis [accumulation of 3,4-dihydroxyphenylalanine (DOPA) after administration of a decarboxylase inhibitor] of dopamine in the median eminence. Intracerebroventricular (icv) injection of histamine produced a dose- and time-dependent increase in plasma prolactin levels but had no effect on DOPA accumulation or DOPAC concentrations in the median eminence. These results indicate that the stimulation of prolactin secretion following icv histamine is not mediated by an inhibition of tuberoinfundibular DA neurons.     

Fire induced stem death in an African acacia is not caused by canopy scorching

Abstract The death of smaller stems of trees due to fire is widespread in savannas. There are currently two hypotheses as to how tree stems avoid stem death; by (i) growing tall and enabling the terminal buds to escape being scorched; and (ii) growing a larger stem diameter and thus being buffered against the heat of the fire. Laboratory‐based tests of these hypotheses on one savanna tree species, Acacia karroo Haynes, support the contention that the important parameter is stem diameter. In addition, anatomical evidence of heat impacts to xylem suggests that damage to the xylem of a stem may play a mechanistic role in causing stem death.     

Neuropeptide Y-induced operant responding for sucrose is not mediated by dopamine

Neuropeptide Y (NPY) induces feeding in previously satiated animals after injection into the hypothalamus, especially the perifornical region (PFH). NPY also appears to have rewarding properties as evidenced by its ability to produce a conditioned place preference following injection into the nucleus accumbens (N.Acc), an effect ostensibly mediated by mesolimbic dopamine (DA). Since the progressive-ratio (PR) operant schedule has also been used to assess an organism’s motivation to respond for rewarding stimuli, we tested the possibility that NPY increases PR responding for sucrose pellets. Adult male rats were injected with NPY (0–235 pmol) bilaterally through cannulae aimed to terminate in the PFH. This produced a dose-dependent increase in the total number of responses made and the number of reinforcers earned. The DA receptor blocker, α-flupenthixol (FLU)(0–0.2 mg/kg intraperitoneally), attenuated both NPY(156 pmol)-induced and drug-free PR responding while having no effect on NPY(156 pmol)-induced free-feeding. FLU injected directly into the N.Acc (0–5 μg) also failed to reduce sucrose free-feeding. These results suggest that distinct reward mechanisms are activated during PFH NPY-induced feeding vs. PR responding, since FLU disrupted the latter but not the former.