Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNAiMet binding to the ribosome

eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA_i^Met) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE₁) and SE₂, was found to stimulate recruitment of Met-tRNA_i^Met in the ternary complex (TC) with eIF2·GTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI₁ and SI₂, reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA_i^Met in the P site for AUG selection.     

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A^*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8×A2.1/K^b mouse-derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81–88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81–88), we found that the heterodimeric CD8αβ coreceptor, but not normally expressed homodimeric CD8αα, is required for tetramer binding and functional redirection of TCR-transduced human T cells. CD8⁺T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 antibody and required more peptide than wild-type (WT) MDM2 TCR⁺ T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) αβ domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expresion, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease. These two authors contributed equally to this work.     

Moderate altitude but not additional endurance training increases markers of oxidative stress in exhaled breath condensate

Oxidative stress occurs at altitude, and physical exertion might enhance this stress. In the present study, we investigated the combined effects of exercise and moderate altitude on redox balance in ten endurance exercising biathletes, and five sedentary volunteers during a 6-week-stay at 2,800 m. As a marker for oxidative stress, hydrogen peroxide (H₂O₂) was analyzed by the biosensor measuring system Ecocheck™, and 8-iso prostaglandin F2α (8-iso PGF2α) was determined by enzyme immunoassay in exhaled breath condensate (EBC). To determine the whole blood antioxidative capacity, we measured reduced glutathione (GSH) enzymatically using Ellman’s reagent. Exercising athletes and sedentary volunteers showed increased levels of oxidative markers at moderate altitude, contrary to our expectations; there was no difference between both groups. Therefore, all subjects’ data were pooled to examine the oxidative stress response exclusively due to altitude exposure. H₂O₂ levels increased at altitude and remained elevated for 3 days after returning to sea level (p ≤ 0.05). On the other hand, 8-iso PGF2α levels showed a tendency to increase at altitude, but declined immediately after returning to sea level (p ≤ 0.001). Hypoxic exposure during the first day at altitude resulted in elevated GSH levels (p ≤ 0.05), that decreased during prolonged sojourn at altitude (p ≤ 0.001). In conclusion, a stay at moderate altitude for up to 6 weeks increases markers of oxidative stress in EBC independent of additional endurance training. Notably, this oxidative stress is still detectable 3 days upon return to sea level.

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The contribution of Max Gassmann and Katja Heinicke who are the senior authors was equivalent.     

Adrenaline-, not somatostatin-induced hyperpolarization is accompanied by a sustained inhibition of insulin secretion in INS-1 cells. Activation of sulphonylurea K ATP + channels is not involved

Adrenaline and somatostatin inhibit insulin secretion via pertussis toxin (PTX)-sensitive mechanisms. Since glucose-stimulated release involves inhibition of ATP-sensitive K⁺ (K⁺ _ATP) channels and activation of Ca²⁺ influx, we took advantage of the glucose-sensitive, insulin-secreting cell line INS-1 to investigate whether inhibitors of insulin release modulate membrane voltage and K⁺ _ATP channel activity in cell-attached patch-clamp experiments. We found that adrenaline, through α₂-adrenoceptors, and somatostatin counteracted glucose-induced depolarization and action potentials. As expected, these effects were mediated via PTX-sensitive G proteins since PTX pretreatment of the cells eliminated the effects of adrenaline and somatostatin on membrane voltage. When INS-1 cells were activated by adding both the K⁺ _ATP channel inhibitor tolbutamide and the adenylyl cyclase activator forskolin, adrenaline and somatostatin still repolarized the plasma membrane. Single-channel measurements in the cell-attached mode revealed that tolbutamide closed a 40 to 70 pS K⁺ channel which was neither reopened by adrenaline nor by somatostatin. In parallel cell preparations, insulin secretion was measured by radioimmunoassay. Insulin release induced by glucose, forskolin and tolbutamide was abolished by adrenaline. In contrast, somatostatin attenuated insulin secretion by only 30%. After comparing the potency of adrenaline and somatostatin on membrane voltage and on insulin secretion, it is concluded that the repolarizing effect of adrenaline on membrane voltage is not sufficient to explain its potent inhibitory effect on insulin secretion. Received: 10 October 1995/Received after revision: 3 January 1996/Accepted: 9 January 1996     

Loss of immunohistochemical E-cadherin expression in colon cancer is not due to structural gene alterations

 E-cadherin, a transmembrane cell adhesion molecule, has been observed to have an altered pattern of immunoreactivity in several types of carcinomas. In lobular breast cancer, loss of immunoreactivity has been shown to be due either to out-of-frame deletions or to nonsense mutations of the E-cadherin gene. We analysed 29 cases of completely resected colon carcinoma with immunohistochemistry using the HEC-D1 antibody. Normal protein expression similar to that in the adjacent nonmalignant mucosa was seen in 6 cases, whereas 23 tumours had reduced or absent E-cadherin expression. In the 8 cases with no expression of E-cadherin revealed by immunohistochemistry, the entire E-cadherin cDNA sequence was analysed. In these cases, sequence analysis failed to reveal any cDNA mutations despite the negative immunohistochemistry. Possible explanations for this discrepancy include regulatory defects in the E-cadherin promoter, abnormalities at the translation or protein processing levels and mutations in other parts of the gene that were not investigated by the cDNA analysis (e.g. intronic sequences), which could play a role in causing abnormal processing of the E-cadherin protein. Received: 14 September 1998 / Accepted: 23 February 1999     

Two types of ω-agatoxin IVA-sensitive Ca2+ channels are coupled to adrenaline and noradrenaline release in bovine adrenal chromaffin cells

 To clarify the role of P-type Ca²⁺ channels in catecholamine release from adrenal chromaffin cells we examined the concentration dependence of the effect of ω-agatoxin IVA on the release both of adrenaline and noradrenaline induced by a K⁺-evoked depolarization. ω-Agatoxin IVA caused a biphasic dose-dependent inhibition of secretion with a high-potency component (IC₅₀<1 nM), responsible for 10–15% of catecholamine release evoked by 70 mM K⁺, and a low-potency component that accounted for about 40% of release, with IC₅₀ values of 57 nM and 48 nM for noradrenaline and adrenaline release, respectively. The release of catecholamines from chromaffin cells was also inhibited dose dependently by ω-conotoxin MVIIC with IC₅₀ values of 182 and 218 nM for noradrenaline and adrenaline release, respectively. The effects of 3 nM ω-agatoxin IVA and 3 μM ω-conotoxin MVIIC were additive, indicating that at the concentrations used the toxins were acting at independent sites, presumably, P- and Q-type Ca²⁺ channels. The blockade of Q-type channels inhibited the release of adrenaline (72 ± 4.1%) significantly more than the release of noradrenaline (50 ± 2.7%), suggesting a higher density or a closer coupling of these channels to exocytosis in adrenergic chromaffin cells. The blockade of P-type channels caused a greater inhibition of catecholamine secretion at low levels of K⁺-evoked depolarization and shorter times of stimulation than that observed at higher levels of stimulation. The contribution of Q-type channels to catecholamine secretion did not change significantly with the intensity of stimulation. The data show that two types of ω-agatoxin IVA-sensitive Ca²⁺ channels are coupled to catecholamine release in chromaffin cells, and that the contribution of P-type channels to secretion is larger at low levels of depolarization. Received: 6 March 1997 / Received after revision and accepted: 28 April 1997     

Translational repression of inducible NO synthase in macrophages by l-arginine depletion is not associated with an increased phosphorylation of eIF2α

In mouse inflammatory macrophages the cytokine-mediated expression of inducible nitric oxide synthase (iNOS) is regulated by the availability of the substrate l-arginine. Following arginine starvation the levels of iNOS mRNA remain unimpaired, whereas the translation of iNOS protein is strikingly downregulated. In the present study we addressed the question, whether arginine-deficient macrophages follow the canonical integrated stress response (ISR) that in other cell types depleted of amino acids was characterized by the accumulation of phosphorylated (i.e. inactive) eukaryotic translation initiation factor-2α (eIF2α), the attenuation of global protein synthesis and the induction of certain stress response target genes. Unexpectedly, resting as well as stimulated inflammatory macrophages constitutively exhibited high levels of phosphorylated eIF2α, which was not further increased upon l-arginine starvation. At the same time, macrophages deprived of l-arginine showed a significant upregulation of the mRNA levels of ISR genes. From these data we conclude that l-arginine deficiency blocks the translation of iNOS and elicits a stress response in macrophages, both of which, however, do not result from an enhanced phosphorylation of eIF2α. Alternative modes of translational repression of iNOS need to be considered.     

The T911C (F304S) substitution in the human ALG6 gene is a common polymorphism and not a causal mutation of CDG-Ic

A T911C (F304S) substitution in the ALG6 gene involved in congenital disorder of glycosylation type Ic (OMIM 603147) has been described. However, whether the F304S substitution is a common polymorphism or a causal mutation remains unclear. We screened for the T911C substitution in the ALG6 gene in 54 unrelated healthy French individuals. We developed a restriction fragment length polymorphism assay with a mutagenic primer introducing a diagnostic DdeI restriction site. We found 23 heterozygotes (42.6%) and 3 homozygous individuals (5.5%). This result indicates that T911C is a common polymorphism with an allele frequency of 27% in a French population and not a causal mutation of congenital disorder of glycosylation type Ic. Received: March 6, 2001 / Accepted: June 1, 2001     

The nuclear envelope of resting C6 glioma cells is able to release and uptake Ca2+ in the absence of chemical stimulation

 Many agonists evoke events in the cell nucleus through the control of Ca²⁺ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca²⁺. However, the release of Ca²⁺ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca²⁺ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca²⁺-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca²⁺ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca²⁺ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca²⁺ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 μM), a specific Ca²⁺-ATPase inhibitor, a long-lasting Ca²⁺ release was observed at the nuclear envelope. Moreover, most of the released Ca²⁺ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca²⁺ store that possesses the ability to discharge and sequestrate Ca²⁺; and (2) the Ca²⁺-releasing channels are present in the inner nuclear membrane. Received: 21 July 1997 / Received after revision: 22 September 1997 / Accepted: 23 September 1997     

Expression of matrix metalloproteinase-1 mRNA related to eosinophilia and interleukin-5 gene expression in head and neck tumour tissue

 In situ hybridization (ISH) of the expression of matrix metalloproteinase-1 (MMP-1) in areas of local invasion of head and neck tumours was performed. Expression of MMP-1 mRNA was found in stromal inflammatory cells adjacent to the tumour nests, apparently in eosinophils. MMP-1 mRNA expression was seen only in cases with moderate or greater stromal eosinophilia. MMP-1 mRNA was detected in benign papilloma and verrucous carcinoma. In squamous cell carcinomas, expression of the gene was detected only in well-differentiated cases. Expression of MMP-1 mRNA was not related to the grade of malignancy, and appeared in stromal eosinophils, suggesting to us that it is involved in a remodelling process in the reaction of the host to tumour invasion. ISH examination of the expression of interleukin-5 (IL-5), the activating factor for eosinophils, was also performed. In the tumour, its expression was consistent with the location of eosinophils, as with MMP-1 mRNA, suggesting an autocrine mechanism. Received: 28 February 1997 / Accepted: 29 April 1997     

Transforming growth factor-beta expression in human retinal pigment epithelial cells is enhanced by Toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis

Retinochoroiditis caused by Toxoplasma gondii infection results in inflammation and necrosis of the retina. We have used human retinal pigment epithelial cultures (HRPE) as an in vitro model to investigate the role of TGF-beta in T. gondii-induced retinochoroiditis. RT-PCR analyses showed enhanced steady state levels of TGF-beta1 and TGF-beta2 mRNA in T. gondii-infected HRPE. Uninfected HRPE secrete TGF-beta1 in a latent form while 10-30% of the secreted TGF-beta2 was in the active form. T. gondii infection induced a significant increase (P < 0.01) in total TGF-beta1 and TGF-beta2 secretion by HRPE. In addition, soluble extracts of T. gondii (ST) stimulated secretion of both TGF-beta1 and TGF-beta2 significantly (P < 0.01). Interestingly, T. gondii infection as well as ST of the parasites completely inhibited secretion of the active form of TGF-beta2. Studies evaluating the effect of TGF-beta on T. gondii replication in HRPE revealed that TGF-beta enhanced parasite replication. The interactions between host retinal cells and T. gondii may play an active role in the pathogenesis of retinochoroiditis.     

Use of monoclonal antibodies to study interleukin -1β-converting enzyme expression: only precursor forms are detected in interleukin-1β-secreting cells

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1β-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1β was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1β cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.     

Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging

The Saccharomyces cerevisiae Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, plays a key role in mitochondrial function, oxidative stress resistance and chronological lifespan. Isc1p functions upstream of the ceramide-activated protein phosphatase Sit4p through the modulation of ceramide levels. Here, we show that both ceramide and loss of Isc1p lead to the activation of Hog1p, the MAPK of the high osmolarity glycerol (HOG) pathway that is functionally related to mammalian p38 and JNK. The hydrogen peroxide sensitivity and premature aging of isc1Δ cells was partially suppressed by HOG1 deletion. Notably, Hog1p activation mediated the mitochondrial dysfunction and catalase A deficiency associated with oxidative stress sensitivity and premature aging of isc1Δ cells. Downstream of Hog1p, Isc1p deficiency activated the cell wall integrity (CWI) pathway. Deletion of the SLT2 gene, which encodes for the MAPK of the CWI pathway, was lethal in isc1Δ cells and this mutant strain was hypersensitive to cell wall stress. However, the phenotypes of isc1Δ cells were not associated with cell wall defects. Our findings support a role for Hog1p in the regulation of mitochondrial function and suggest that constitutive activation of Hog1p is deleterious for isc1Δ cells under oxidative stress conditions and during chronological aging.     

B cell early response gene expression coupled to B cell receptor,CD40 and interleukin-4 receptor co-stimulation: evidence for a role of the egr-2/krox20 transcription factor in B cell proliferation

B lymphocytes are activated following antigen stimulation of the B cell receptor but require co-stimulation with accessory molecules provided by interleukin (IL)-4/CD40 ligand for cell cycle progression and proliferation. By analyzing a panel of 11 early response genes induced by cross-linking of surface immunoglobulin, we show that CD40 signaling alone induces only 2 genes, c-myc together with an anonymous gene, 3L3, and that these are distinct from the set of genes induced in response to IL-4. Co-stimulation with the proliferative combination of anti-μ, IL-4 + CD40 signaling led to a fourfold enhancement of egr-2/krox20 expression over that seen with anti-μ alone. Egr-2 expression/activity was selectively inhibited by the immunosuppressive drug cyclosporin A, and antisense oligonucleotide blockade of Egr-2 activity elicited a dose-dependent inhibition of B cell proliferation. Taken together, these observations show that the early gene regulatory programs coupled to different surface receptors on B cells are largely distinct from each other, but that certain genes, exemplified by egr-2, may represent a point of convergence in the integration of different signaling pathways into the B cell proliferative response.     

MD‐2 is involved in the stimulation of matrix metalloproteinase‐1 expression by interferon‐γ and high glucose in mononuclear cells – a potential role of MD‐2 in Toll‐like receptor 4‐independent signalling

We reported recently that treatment of diabetic apolipoprotein E‐deficient mice with the Toll‐like receptor 4 (TLR4) antagonist Rs‐LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs‐LPS antagonizes TLR4 by targeting TLR4 co‐receptor MD‐2, this finding indicates that MD‐2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD‐2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon‐γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD‐2 is involved in IFN‐γ and high‐glucose‐regulated gene expression in mononuclear cells. Results showed that IFN‐γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP‐1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs‐LPS inhibited the MMP‐1 stimulation. To provide more evidence for a role of MD‐2 in IFN‐γ‐stimulated MMP‐1, studies using antibodies and small interfering RNA demonstrated that MD‐2 blockade or knockdown attenuated the effect of IFN‐γ on MMP‐1. Furthermore, studies using PCR arrays showed that MD‐2 blockade had a similar effect as IFN‐γ receptor blockade on the inhibition of IFN‐γ‐stimulated pro‐inflammatory molecules. Although these findings indicate the involvement of MD‐2 in IFN‐γ signalling, we also observed that MD‐2 was up‐regulated by IFN‐γ and high glucose. We found that MD‐2 up‐regulation by IFN‐γ played an essential role in the synergistic effect of IFN‐γ and LPS on MMP‐1 expression. Taken together, these findings indicate that MD‐2 is involved in IFN‐γ signalling and IFN‐γ‐augmented MMP‐1 up‐regulation by LPS.     

Primary over‐expression of AβPP in muscle does not lead to the development of inclusion body myositis in a new lineage of the MCK‐AβPP transgenic mouse

The aim of this study is to determine whether primary over‐expression of AβPP in skeletal muscle results in the development of features of inclusion body myositis (IBM) in a new lineage of the MCK‐AβPP transgenic mouse. Quantitative histological, immunohistochemical and western blotting studies were performed on muscles from 3 to 18 month old transgenic and wild‐type C57BL6/SJL mice. Electron microscopy was also performed on muscle sections from selected animals. Although western blotting confirmed that there was over‐expression of full length AβPP in transgenic mouse muscles, deposition of amyloid‐β and fibrillar amyloid could not be demonstrated histochemically or with electron microscopy. Additionally, other changes typical of IBM such as rimmed vacuoles, cytochrome C oxidase‐deficient fibres, upregulation of MHC antigens, lymphocytic inflammatory infiltration and T cell fibre invasion were absent. The most prominent finding in both transgenic and wild‐type animals was the presence of tubular aggregates which was age‐related and largely restricted to male animals. Expression of full length AβPP in this MCK‐AβPP mouse lineage did not reach the levels required for immunodetection or deposition of amyloid‐β as in the original transgenic strains, and was not associated with the development of pathological features of IBM. These negative results emphasise the potential pitfalls of re‐deriving transgenic mouse strains in different laboratories.