Structure of the human melanin concentrating hormone mRNA.

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide which induces skin paling and may be involved in the control of the pituitary adrenal axis in teleost fishes. We have recently cloned and characterized the salmon and rat MCH mRNAs and we report in the present paper the cloning and sequencing of the human counterpart. The deduced human MCH (hMCH) precursor is 165 amino acids long and as for rat and salmon, encodes the MCH peptide at the C-terminus. The human and rat MCH precursors are very similar to one another but differ extensively from the salmon counterpart. Strong sequence conservation was found in the regions of mammalian prohormones encoding the novel putative neuropeptides named NGE and NEI which we had originally identified in the rat MCH precursor. Furthermore, sequence identities, with perhaps functional implications, were found among the MCH, human ANF, and aplysia peptide A hormone precursors.

Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide first isolated from fish and rats. MCH may be involved in the control of the hypothalamic-pituitary-adrenocortical axis and, more generally, of specific goal-oriented behaviors and homeostatic functions in mammals. In this paper we examine 1) the cellular distribution of MCH gene transcripts in the rat central nervous system, 2) the changes in neuronal expression of MCH mRNA during rat development, and 3) the effects of stress and hormonal stimuli on rat MCH (rMCH) gene activity. Northern blot analysis and in situ hybridization histochemistry show that mature rMCH mRNA (1.0 kilobase) is very abundant in the zona incerta and the dorsolateral hypothalamus. While this is in agreement with previous peptide mapping by immunohistochemical techniques, a surprising new result is that a few clusters of rMCH mRNA-containing cells are found outside the hypothalamus, in the olfactory tubercle and the pontine tegmentum. Developmentally, rMCH mRNA is detected on embryonic day 18; its level increases gradually during early postnatal life and rises abruptly at weaning to reach a constant value in adult rats. In addition, striking variations in rMCH mRNA length occur during postnatal development and are found to be variations in the polyadenylate tail. Interestingly, this structural modification appears to be independent of the increase in rMCH mRNA levels. The regulation of rMCH mRNA expression by glucocorticoids and chronic stress is examined by Northern blot analysis. Chronic intermittent footshock stress causes a 58% or 29% decrease in rMCH mRNA content in the whole hypothalamus after a 1- or 3-day regimen, respectively. In contrast, the rMCH mRNA level returns to normal after a 7-day regimen. Two weeks after adrenalectomy (ADX) the whole hypothalamus rMCH mRNA content decreases 2.5-fold, but rises close to the control value 3 weeks after ADX. Dexamethasone administration 2 weeks after ADX not only reverses the fall in rMCH mRNA, it even provokes a slight increase (123% of control). No change in rMCH mRNA length is observed after chronic stress or ADX and dexamethasone injection. These results provide evidence for a negative regulation of rMCH gene expression by stress and suggest a major role for glucocorticoids in a positive feedback control of rMCH gene activity.

The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus.

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide, first isolated from salmon pituitary glands, which regulates melanin dispersion in the skin and perhaps the activity of the pituitary-adrenal axis in teleost fish. We have recently purified and characterized rat MCH (rMCH) and report here the cloning and sequencing of specific MCH cDNA isolated from a rat hypothalamic library. The sequence of rMCH found by DNA sequencing confirms the sequence deduced from the purified peptide. rMCH is located at the C-terminus of a protein precursor of 165 amino acid residues. Comparison of the amino acid sequence of prepro-MCH and that of the Aplysia peptide-A prohormone suggests that these proteins as well as other precursors may be evolutionarily related. Besides rMCH, two putative neuropeptides, termed NGE and NEI, might be generated from the same precursor. The rMCH precursor shared sequence identities with human GH-releasing factor and mammalian CRF in the regions encoding NGE and NEI. By immunohistochemical studies we have established that the amidated C-terminus of NEI is recognized by some alpha MSH and rat CRF antisera and that the C-terminal portion of NGE is responsible for the cross-reactivity revealed with one hGRF-(1-37) antiserum. Our results explain the staining of a discrete population of dorso-lateral hypothalamic neurons by heretofore seemingly unrelated antisera and provide evidence for the production of multiple novel neuropeptides from a common precursor.

The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization.

In addition to a nonadecapeptide homologous to the teleost melanin-concentrating hormone (MCH), the amino acid sequence predicted from a rat prepro-MCH (ppMCH) cDNA suggested that at least one (neuropeptide EI, or NEI), and possibly a second (NGE), additional neuropeptide may be encoded by this precursor. Cross-reactivity with epitopes of NEI or NGE can account for reported localization of alpha-MSH, rat CRF, and human GRF in rat dorsolateral hypothalamic neurons. We have used antisera raised against rat MCH and NEI in immunohistochemical studies at the light and electron microscopic levels, along with hybridization histochemical localization of ppMCH mRNA, to define the organization of this system. As expected, ppMCH mRNA is prominently expressed in cells in the lateral hypothalamic area and zona incerta. The MCH and NEI peptides were extensively colocalized in neurons in both of these areas. In addition, smaller cell groups in the olfactory tubercle and pontine tegmentum were also positively hybridized for ppMCH mRNA and immunostained for MCH and NEI. Fibers stained for MCH and NEI were similarly, and very broadly, distributed throughout the central nervous system in patterns that generally conformed with known projection fields of the lateral hypothalamic area and zona incerta. A differential distribution was seen in at least one region, the interanterodorsal nucleus of the thalamus, which contained a prominent terminal field stained for MCH but not NEI. At the electron microscopic level, MCH-stained perikarya displayed a prominent staining associated with the Golgi apparatus; this was not encountered in NEI-stained cells. Both peptides were distributed similarly in terminals in the lateral hypothalamic area and median eminence, with staining associated principally with dense-cored vesicles. The results suggest that ppMCH-derived peptides may serve as neurotransmitters or modulators of prominence in a surprisingly expansive projection field of incerto-hypothalamic neurons. The terminal distributions of this system seem most compatible with functional roles in generalized arousal and sensorimotor integration, processes previously implicated as being subject to modulation by the lateral hypothalamic area.

Differential regulation of melanin-concentrating hormone gene expression in distinct hypothalamic areas under osmotic stimulation in rat.

Melanin-concentrating hormone and associated peptides represent a novel peptide neuronal system that may be involved in the control of water homeostasis in mammals. We have examined the effect of 24 h dehydration or salt-loading over a period of six days, on melanin-concentrating hormone messenger RNA levels in rat brains by using complementary methods of Northern blotting and in situ hybridization histochemistry. In response to one to six day salt-loading regimen, hypothalamic melanin-concentrating hormone messenger RNA content in male or female rats decreased by two to three-fold. Levels of melanin-concentrating hormone messenger RNA in the hypothalamus were also dramatically decreased following dehydration in female rats whereas contrasting responses were noted in male rats. In addition, no significant variation in the low levels of melanin-concentrating hormone gene transcripts in medulla pons and cortex was found after osmotic stimulus. In agreement with Northern blot data, in situ hybridization studies revealed that the majority of the melanin-concentrating hormone-expressing neurons in the anterior part of the lateral hypothalamus of dehydrated or salt-loaded rats expressed lower amounts of melanin-concentrating hormone messenger RNAs than those found in control rats. Interestingly, less variation was found in the posterior part of the lateral hypothalamus. Furthermore few clusters of cells, located in zona incerta and near the internal capsula and fornix, increased their contents in melanin-concentrating hormone messenger RNA in salt-loaded but not in dehydrated rats suggesting that melanin-concentrating hormone gene expression may be regulated differently by various osmotic stimuli. Finally, diurnal variations in melanin-concentrating hormone messenger RNA contents were observed in normal and dehydrated rats with highest levels around 22.00 h and lowest levels during daylight hours. However, the up-regulation of melanin-concentrating hormone gene activity at night was found lower in dehydrated rats than in control animals suggesting that osmotic stress may interfere with the generation of the diurnal pattern of melanin-concentrating hormone messenger RNA expression. Altogether, our results indicate that osmotic stimulations lead to a selective and conspicuous inhibition of melanin-concentrating hormone gene activity in the whole hypothalamus of rat. We suggest that the melanin-concentrating hormone neuronal system plays an important role in integration processes relative to nocturnal regulation of water homeostasis and drinking behavior.

Melanin-concentrating hormone is a potent anorectic peptide regulated by food-deprivation and glucopenia in the rat.

Melanin-concentrating hormone is a cyclic nonadecapeptide that is produced almost exclusively in neurons of the lateral hypothalamus and sub zona incerta areas while fibers are widespread in the rat brain. Such a localization strongly suggests that this peptide might participate as neurotransmitter/neuromodulator in the control of feeding behavior. In this study we examined first the influence of rat melanin-concentrating hormone on feeding behavior at different times either in the light or in the dark period (light off at 18.00 h) of the day in fed Wistar rats. Intracerebroventricular injection of rat melanin-concentrating hormone (1-100 ng per rat) at 18.00 h reduced food consumption as early as 2 h after injection and for the next 24 h. In addition, similar anorectic effect was noted after bilateral administration of 1 ng melanin-concentrating hormone into the lateral hypothalamic area at 11.30 h but not at 16.30 h. These findings strongly suggest that rat melanin-concentrating hormone may exert inhibitory control over food intake behavior depending on the circadian rhythm. Second, we investigated the modifications induced by food deprivation/refeeding on melanin-concentrating hormone messenger RNA levels in Wistar rats. Total RNA was isolated from whole hypothalamic dissections and the contents of melanin-concentrating hormone messenger RNA, beta-actin messenger RNA (taken as sample control) and neuropeptide Y messenger RNA (taken as control of food-deprivation paradigms) were assessed by using northern blotting. The time-course of messenger RNA expression was determined in groups of rats deprived for 24, 48 and 72 h and revealed a three-fold induction of melanin-concentrating hormone messenger RNA by 24 and 48 h, with reduced increase at 72 h. As expected, the same treatment led to a three-fold increase in neuropeptide Y messenger RNA content by 48 and 72 h. Refeeding groups of animals for up to 72 h after 24 h of food deprivation resulted in full restoration of melanin-concentrating hormone messenger RNA levels by 24 h. Strikingly, a large range of variations in melanin-concentrating hormone messenger RNA content between individuals was observed in food-deprived versus controls or refed rats suggesting that genetic or environmental factors may alter response in melanin-concentrating hormone gene activity after food deprivation. Finally, we investigated the effects of short-term glucoprivation induced by intraperitoneal administration of either 2-deoxy-D-glucose or insulin on melanin-concentrating hormone messenger RNA expression. A transitory increase in melanin-concentrating hormone messenger RNA content was noted 1 h after 2-deoxy-D-glucose injection while melanin-concentrating hormone messenger RNA levels rose two-fold only 5 h after insulin treatment. These results indicate that 2-deoxy-D-glucose and insulin activate melanin-concentrating hormone gene expression through likely distinct regulatory pathways.

Lithium increases melanin-concentrating hormone mRNA stability and inhibits tyrosine hydroxylase gene expression in PC12 cells.

Melanin-concentrating hormone (MCH) is a cyclic peptide involved in the regulation of food-intake behaviour and stress response in mammals. Expression of the MCH gene predominates in hypothalamic neurons. Mechanisms governing the regulation of expression of MCH gene in established cell lines were not explored yet. Here, we analysed the actions of nerve growth factor (NGF), dexamethasone, forskolin and lithium on MCH mRNA levels in the PC12 pheochromocytoma cell line. We compared them with those observed on tyrosine hydroxylase (TH) mRNA, constitutively expressed in PC12 cells, and neurotensin (NT) mRNA, taken as a control. In untreated cells, MCH RNA species of high molecular weight were found. Exposure of cells at a combination of NGF and lithium resulted in decreased expression of these MCH RNAs and in the transient production of mature MCH mRNA. Strikingly, after short exposure of PC12 cells to NGF, lithium per se elicited a marked increase in MCH mRNA levels whilst it exerted a potent inhibitory action on TH mRNA expression. Detailed investigations revealed that lithium enhanced MCH mRNA expression through post-transcriptional mechanisms whereas it regulated TH gene expression mainly at the level of transcription. These results demonstrate that lithium, an agent widely used for treatment of manic depressive illness, can exert an opposite effect on MCH and TH mRNA production in PC12 cells. The MCH gene system in NGF-treated PC12 cells provides a good opportunity for studying the effect of lithium on gene expression at post-transcriptional levels in a neuron-like cellular model.

Neuropeptide-E-I antagonizes the action of melanin-concentrating hormone on stress-induced release of adrenocorticotropin in the rat.

The physiological role of melanin-concentrating hormone (MCH) in mammals is still very elusive, but this peptide might participate in the central control of the hypothalamopituitary adrenal (HPA) axis during adaptation to stress. Cloning and sequencing of the rat MCH (rMCH) cDNA revealed the existence of additional peptides encoded into the MCH precursor. Among these peptides, neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI) is co-processed and secreted with MCH in rat hypothalamus. In the present work we examined: (1) The pattern of rMCH mRNA expression during the light and dark conditions in the rat hypothalamus and (2) The effect of intracerebroventricular (ICV) injections of rMCH and NEI in the control of basal or ether stress-modified release of corticotropin (ACTH), prolactin (PRL) and growth hormone (GH) secretion in vivo in light-on and light-off conditions. Our data indicate that rMCH mRNA levels do not change during the light-on period, but increase after the onset of darkness. Either alone or co-administered, rMCH and NEI do not modify basal secretion of GH and PRL at any time tested nor do they alter ether stress-induced changes in these two hormonal secretions. At the end of the light on period corresponding to the peak of the circadian rhythm in ACTH, administration of rMCH but not NEI leads to a decrease in ACTH levels while MCH is not effective during the light off period of the cycle (i.e. when basal ACTH levels are already low). Using a moderate ether induced stress, ACTH levels are only stimulated during the dark phase of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

The melanin-concentrating hormone receptors: neuronal and non-neuronal functions.

Melanin-concentrating hormone (MCH) is a cyclic peptide highly conserved in vertebrates and was originally identified as a skin-paling factor in Teleosts. In fishes, MCH also participates in the regulation of the stress-response and feeding behaviour. Mammalian MCH is a hypothalamic neuropeptide that displays multiple functions, mostly controlling feeding behaviour and energy homeostasis. Transgenic mouse models and pharmacological studies have shown the importance of the MCH system as a potential target in the treatment of appetite disorders and obesity as well as anxiety and psychiatric diseases. Two G-protein-coupled receptors (GPCRs) binding MCH have been characterized so far. The first, named MCH-R1 and also called SLC1, was identified through reverse pharmacology strategies by several groups as a cognate receptor of MCH. This receptor is expressed at high levels in many brain areas of rodents and primates and is also expressed in peripheral organs, albeit at a lower rate. A second receptor, designated MCH-R2, exhibited 38% identity to MCH-R1 and was identified by sequence analysis of the human genome. Interestingly, although MCH-R2 orthologues were also found in fishes, dogs, ferrets and non-human primates, this MCH receptor gene appeared either lacking or non-functional in rodents and lagomorphs. Both receptors are class I GPCRs, whose main roles are to mediate the actions of peptides and neurotransmitters in the central nervous system. However, examples of action of MCH on neuronal and non-neuronal cells are emerging that illustrate novel MCH functions. In particular, the functionality of endogenously expressed MCH-R1 has been explored in human neuroblastoma cells, SK-N-SH and SH-SY5Y cells, and in non-neuronal cell types such as the ependymocytes. Indeed, we have identified mitogen-activated protein kinase (MAPK)-dependent or calcium-dependent signalling cascades that ultimately contributed to neurite outgrowth in neuroblastoma cells or to modulation of ciliary beating in ependymal cells. The putative role of MCH on cellular shaping and plasticity on one side and volume transmission on the other must be now considered.

Structure and regulation of the mouse melanin-concentrating hormone mRNA and gene.

Melanin-concentrating hormone (MCH) and associated peptides, designated NEI and NGE, are predominantly expressed in hypothalamic neurons which project widely throughout the mammalian brain. These peptides might be involved as neuromodulators in the control of goal-directed behaviors, the integration of the stress response, and/or the regulation of arousal in general. In vivo studies of this peptidic system using a transgenic mouse model call for information about the structure and regulation of the mouse MCH (mMCH) gene. One complementary DNA (cDNA) for mouse prepro-MCH was isolated from a brain library by using a rat MCH cDNA as probe. This cDNA contains an open reading frame coding for a 165-amino acid precursor that displays about 90% sequence identity with rat and human prepro-MCHs. Most of the structural portion of the mMCH gene was cloned and characterized using the polymerase chain reaction (PCR). Strong conservation in exon-intron organization and primary sequences was found among the mouse, rat, and human genes, suggesting that coding and noncoding regions have important biological functions. Developmentally regulated expression of mMCH gene in mouse hypothalamus was examined by Northern blot hybridization. Up to 10-fold changes in the relative mMCH mRNA contents were observed during postnatal development, characterized by a peak at the weaning period. Moreover, striking variations in mMCH mRNA length, due to a poly(A) tail, were revealed during postnatal life. Tissue distribution of mMCH gene transcripts was investigated by means of the PCR and Northern blot procedures. Expression of the mMCH gene was revealed in heart, intestine, spleen, and testis and was found to be regulated in a developmentally programmed manner. Strikingly, short as well as long mMCH RNA species were identified at the periphery. Taken together, our data indicate that both transcriptional and post-transcriptional mechanisms regulate the expression of the MCH gene in mouse brain and at the periphery.

The AROM gene, spliced mRNAs encoding new DNA/RNA-binding proteins are transcribed from the opposite strand of the melanin-concentrating hormone gene in mammals.

Melanin-concentrating hormone (MCH) mRNA expression is induced by nerve growth factor and lithium in PC12 cells, whereas three large MCH RNA species are found in untreated cells. In this study, we investigated the structures, regulations of expression, and putative functions of these transcripts. Northern blot, rapid amplification of cDNA ends-polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and sequencing experiments demonstrated that they are antisense RNAs complementary to the MCH gene. Two classes of antisense RNAs could be discriminated as follows: 1) non-coding unspliced RNAs that overlap mainly the coding part of the MCH gene; 2) spliced variant mRNAs complementary to the 3'-flanking end of the MCH gene and that encode putative proteins containing DNA/RNA binding domains. We named this new transcriptional unit AROM for antisense-RNA-overlapping-MCH gene. Spliced variant AROM mRNAs are expressed in a broad range of rat organs. Western blot and immunohistochemistry experiments revealed several proteins with cytoplasmic but also nuclear localization in PC12 cells. Time course studies during nerve growth factor and lithium treatment of PC12 cells indicated a reciprocal regulation of the MCH and AROM gene transcripts, reflected also at the level of AROM proteins. The major translational product is a 64-kDa protein (AROM-p64). Recombinant AROM-p64 displayed high binding to single-stranded DNA and poly(A) homopolymers suggesting that this protein could play a role in mRNA maturation/metabolism.

An improved electrotransfection method using square shaped electric impulsions.

Transfection of DNA into non adherent cells can be achieved by electropermeation. Previously published results, partially successful, were obtained using exponential decaying electric impulsions. However, one limitation of this technique has been the damaging effect of this type of impulsions resulting in poor cell recovery. We report hereby the electropermeation of human lymphoblastoid cell lines using a commercially available electropulsator delivering repeated, short, high voltage, square shaped, electric pulses. The parameters of transfection have been optimized using the "Lucifer Yellow Permeation Assay". With the optimum electric parameters, virtually all the cells were permeated and at least 70% survived the shocking conditions. Both transient expression and permanent integration and expression of DNA was observed.

[Decrease in the transforming action of Rous sarcoma virus produced by avian cells grown in the presence of 5'-deoxy-5'-S-isobutyladenosine (SIBA)]. / Diminution de l'action transformante du virus de sarcome de Rous produit par des cellules aviaires cultivées en présence de 5'-désoxy-5'-S-isobutyladénosine SIBA.

Treatment of Rous Sarcoma virus transformed chick embryo fibroblasts with 1 mM 5'-deoxy-5'-S-isobutyladenosine for 24 hrs. leads to the inhibition of transforming virus production. A kinetic analysis of the inhibition of active virion production revealed that the effect of the drug was time and concentration dependent. After 24 hrs. with 1 mM SIBA, the production of transforming virus was inhibited 165 fold. However, under these conditions there was only a 2 fold inhibition in viral particle production. Thus, these viral particles were either non infective (non adsorbed on cell membrane) or non transforming. The majority of viral particles produced by cells cultured with the drug have a decreased density. Analysis of these virions showed a decrease of protein P19 and an accumulation of proteins with high molecular weight.

Acute and chronic administration of melanin-concentrating hormone enhances food intake and body weight in Wistar and Sprague-Dawley rats.

AIM: Although melanin-concentrating hormone (MCH) is believed to be an important regulator of feeding behavior, both its acute and chronic effects on food intake as well as its interaction with other brain peptides involved in the control of appetite remain unclear. Therefore, the acute effects of MCH on food intake and the chronic effect of MCH on food intake and the gene expression of various hypothalamic peptides involved in the control of appetite were studied in rats. METHODS AND RESULTS: Either the acute or the continuous intraventricular infusion of MCH for 12 days stimulated feeding in both Wistar or Sprague-Dawley rats. Removal of the hypothalamus at the end of the chronic infusion studies allowed measurement of the expression of mRNAs encoding for MCH, neuropeptide Y (NPY), orexin, agouti gene-related peptide, cocaine and amphetamine-related transcript and neurotensin-neuropeptides involved in the control of appetite. Chronic intraventricular infusion of MCH activated only NPY mRNA synthesis in Sprague-Dawley rats. The increase in food intake in response to MCH in Sprague-Dawley rats did not appear to be due to the release of NPY since combination studies demonstrated consistently additive effects of the two peptides on food intake at maximum or near maximum doses. CONCLUSIONS: These results strongly suggest that MCH is an orexigenic peptide involved in the control of both short- and long term food intake in satiated rats and further indicate that the MCH pathway is a possible target for the control of food intake and obesity.

Structure and expression of the variant melanin-concentrating hormone genes: only PMCHL1 is transcribed in the developing human brain and encodes a putative protein.

PMCHL1 and PMCHL2 are two copies of the so-called variant melanin-concentrating hormone (MCH) gene that are located, respectively, on human chromosome 5p14 and 5q13 and that emerged recently during primate evolution. They correspond to a 5'-end truncated version of the MCH gene mapped on chromosome 12q23 and encoding a neuropeptide precursor. The gene organization and regulation of the expression of the variant MCH genes in the human brain are the central issues we investigated. First, the structure and fine chromosomal mapping of the 5p and 5q variant MCH genes were established. These revealed several point mutations and length variations of one CA/TA repeat which allow discrimination between each copy. Using a combination of RACE-PCR, RT-PCR, and sequencing analysis, we provided strong evidence for the expression of the PMCHL1 gene but not the PMCHL2 gene in the human fetal, newborn, and adult brains. Sense, potentially coding, RNAs, as well as noncoding antisense RNAs, were identified and displayed a region-specific expression in the human brain. Strikingly, sense unspliced RNAs of the PMCHL1 gene carried a novel open reading frame and may produce an NLS-containing protein of 8 kDa named VMCH-p8. These transcripts were translated in vitro and in transfected COS cells. Therefore, the PMCHL1 gene provides a unique example of the generation of a gene in the Hominoidae lineage which is specifically transcribed in the developing human brain and has the capacity to be translated into a putative novel protein.

Increased resistance to non-MHC-restricted cytotoxicity related to HLA A, B expression. Direct demonstration using beta 2-microglobulin-transfected Daudi cells.

Experiments in several laboratories have shown that target susceptibility to NK and lymphokine-activated killer (LAK) cytotoxicity is inversely correlated with the target expression of HLA Class I molecules. We present the first direct evidence, obtained by gene transfection, that target cell HLA, A, B expression increases the resistance to the "so-called" non-MHC-restricted cytotoxicity. We have co-transfected, by electroporation, the human beta 2-microglobulin gene and the gene carrying the resistance to geneticin into Daudi cell line. Geneticin selection in culture followed by FACS sorting on the basis of strong positivity with the mAb W6/32 (which is specific for the HLA class I H chain associated to beta 2-microglobulin) have led to the establishment of a HLA+ Daudi cell line permanently expressing HLA A10, A11, and B17 molecules. Studies were performed in vitro to evaluate the susceptibility of these cells to either NK and LAK cytotoxicity. The HLA class I+ Daudi cells exhibit an increased resistance to killing by non-MHC-restricted killer cells (both NK and LAK) as compared with their HLA-Daudi counterpart.

Emergence of a brain-expressed variant melanin-concentrating hormone gene during higher primate evolution: a gene "in search of a function".

Two related but distinct melanin-concentrating hormone (MCH) gene systems, i.e., the authentic and variant genes, have been characterized in the human, while only a single MCH gene has been found in the rat. We previously established that the variant gene corresponds to exon-I-deleted copies of the authentic gene mapped on chromosomes 5 and 12, respectively. In this report, we examined the expression of the authentic and variant MCH genes in the human brain. Mature mRNAs of the authentic MCH gene appeared to be predominantly expressed in the hypothalamus, whereas putative unprocessed transcripts of the variant MCH gene were found in other brain areas but not in the hypothalamus. Several products of the variant MCH gene were identified by RACE-PCR in the fetal human brain. One of these transcripts encoded a putative protein of 72 amino acids, while another transcript may potentially generate a protein of 35 amino acids. Thereafter, we explored the question of MCH gene transposition during Primate evolution. Southern blotting, PCR analyses using several genomic DNAs of Primates, and in situ hybridization on metaphase chromosomes led us to define at least three types of genetic events associated with the emergence of the variant MCH gene: (1) translocation of an exon II-exon III copy of the authentic MCH gene onto the equivalent of the human chromosome 5p arm of Anthropoidea ancestors (between 25 and 55 MYA); (2) exon II truncation and mutations before divergence of the Hylobatidae (about 15 MYA); and (3) duplication of the variant gene on the equivalent of the human chromosome 5p and 5q arms in the Hominidae, i.e., 5-10 MYA. Taken together, these results support the hypothesis that transposition/gene rearrangement processes could underlie the evolutionary emergence of new MCH-related genes expressed differentially in the brains of higher Primates, illustrating the concept of genes "in search of function" instead of true "pseudogenes."

Albumin and alpha-fetoprotein gene expression in various nonhepatic rat tissues.

Albumin and alpha-fetoprotein (AFP), two major serum proteins, are synthesized predominantly in the liver and yolk sac of mammals. In the present paper we report on the developmental expression of the corresponding genes in nonhepatic rat tissues. Significant quantities of mature albumin and AFP mRNAs were revealed in kidney, pancreas, heart, and lung of fetal and/or newborn rats using dot blot and Northern blot assays. Very low levels of these mRNA sequences were also detected in adult kidney and pancreas using sensitive RNA-cDNA solution hybridization assays. In situ hybridization analysis revealed that the albumin and AFP gene transcripts are present in the tubular cells of the 20-day-old fetal kidney. In order to elucidate further the mechanisms governing this expression, we studied the chromatin structure and methylation pattern in the 5'-end of these two genes. A faint band, corresponding to a specific DNase I-hypersensitive site upstream from the albumin gene, was detected in the fetal and neonatal kidney nuclei but not in adult kidney. For both genes, a site CG, demethylation of which is correlated with expression in liver and hepatoma cell lines, is highly methylated in fetal kidney even though AFP and albumin genes are expressed. Taken together, these results show the presence of a cell population in the rat kidney that actively transcribes both the albumin and AFP genes. The expression of these genes may be mediated by mechanisms differing in at least some steps from those exerted in the liver.