HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress

Orphanin FQ (OFQ, Nociceptin) is a recently discovered 17-amino acid neuropeptide that is structurally related to the opioid peptides but does not bind opioid receptors. OFQ has been proposed to act as an anti-opioid peptide, but its widespread sites of action in the brain suggest that it may have more general functions. Here we show that OFQ plays an important role in higher brain functions because it can act as an anxiolytic to attenuate the behavioral inhibition of animals acutely exposed to stressful/anxiogenic environmental conditions. OFQ anxiolytic-like effects were consistent across several behavioral paradigms generating different types of anxiety states in animals (light-dark preference, elevated plus-maze, exploratory behavior of an unfamiliar environment, pharmacological anxiogenesis, operant conflict) and were observed at low nonsedating doses (0.1–3 nmol, intracerebroventricular). Like conventional anxiolytics, OFQ interfered with regular sensorimotor function at high doses (>3 nmol). Our results show that an important role of OFQ is to act as an endogenous regulator of acute anxiety responses. OFQ, probably in concert with other major neuropeptides, exerts a modulatory role on the central integration of stressful stimuli and, thereby, may modulate anxiety states generated by acute stress.

The Saccharomyces cerevisiae Homologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

Yeast Las17 protein is homologous to the Wiskott–Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Δ ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Δ cells showed that receptor-mediated internalization of α factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

Transport of Sterols to the Plasma Membrane of Leek Seedlings1

To investigate the intracellular transport of sterols in etiolated leek (Allium porrum L.) seedlings, in vivo pulse-chase experiments with [1-14C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3β-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3β-ol, 24-methyl-cholest-5-en-3β-ol, (24S)-24-ethylcholesta-5,22E-dien-3β-ol, and stigmasta-5,24(241)Z-dien-3β-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12°C) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus.

N-Acylethanolamines: Formation and Molecular Composition of a New Class of Plant Lipids1

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120–126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [14C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [14C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133–142) raises the possibility that a similar mechanism may operate in plant cells.

Stress Induction of Mitochondrial Formate Dehydrogenase in Potato Leaves1

In higher plants formate dehydrogenase (FDH, EC 1.2.1.2.) is a mitochondrial, NAD-dependent enzyme. We previously reported that in potato (Solanum tuberosum L.) FDH expression is high in tubers but low in green leaves. Here we show that in isolated tuber mitochondria FDH is involved in formate-dependent O2 uptake coupled to ATP synthesis. The effects of various environmental and chemical factors on FDH expression in leaves were tested using the mitochondrial serine hydroxymethyltransferase as a control. The abundance of FDH transcripts is strongly increased under various stresses, whereas serine hydroxymethyltransferase transcripts decline. The application of formate to leaves strongly enhances FDH expression, suggesting that it might be the signal for FDH induction. Our experiments using glycolytic products suggest that glycolysis may play an important role in formate synthesis in leaves in the dark and during hypoxia, and in tubers. Of particular interest is the dramatic accumulation of FDH transcripts after spraying methanol on leaves, as this compound is known to increase the yields of C3 plants. In addition, although the steady-state levels of FDH transcript increase very quickly in response to stress, protein accumulation is much slower, but can eventually reach the same levels in leaves as in tubers.

A simple p53 functional assay for screening cell lines, blood, and tumors.

Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors. We have developed a simple functional assay for p53 mutation in which human p53 expressed in Saccharomyces cerevisiae activates transcription of the ADE2 gene. Consequently, yeast colonies containing wild-type p53 are white and colonies containing mutant p53 are red. Since this assay tests the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations. By combining this approach with gap repair techniques in which unpurified p53 reverse transcription-PCR products are cloned by homologous recombination in vivo it is possible to screen large numbers of samples and multiple clones per sample for biologically important mutations. This means that mutations can be detected in tumor specimens contaminated with large amounts of normal tissue. In addition, the assay detects temperature-sensitive mutants, which give pink colonies. We show here that this form of p53 functional assay can be used rapidly to detect germline mutations in blood samples, somatic mutations in tumors, and mutations in cell lines.