The present invention relates to novel splicing variants of the serotoninergic receptor 5-HT4 in humans.
Serotonin (5-hydroxytryptamine or 5-HT) is a neurotransmitter which is located in the central and peripheral nervous system of vertebrates, where it exerts various physiological roles mediated by different receptor subtypes (Saxena, 1995). The receptors 5-HT4 represent a member of the family of receptors with seven transmembrane (7TM) domains coupled to a G protein which is positively coupled to adenylate cyclase (Hedge and Eglen, 1996). The receptors 5-HT4 are expressed in a wide variety of tissues, including the human brain and the rodent brain (Eglen et al., 1995), the human, dog, pig and rodent gastro-intestinal tract, and the pig and human heart (Hedge and Eglen, 1996). In the mammalian brain, the receptors 5-HT4 contribute to dopamine secretion (Bonhomme et al., 1995) and regulate learning and long-term memory via the modification of acetylcholine release (Marchetti-Gauthier et al., 1997). In the peripheral tissues, the receptors 5-HT4 have proven to regulate gastro-intestinal tract motility, intestinal electrolyte secretion, adrenal secretion of corticosteroides, bladder contraction and atrium contractility (Edge and Eglen, 1996).
The receptors 5-HT4 are involved in a wide variety of central and peripheral disorders, including cardiac arrhythmias (Kaumann, 1994) and neurodegenerative disorders (Reynolds et al., 1996; Wong et al., 1996). In addition, the development of receptor 5-HT4 agonists and antagonists may have therapeutic applications in the central nervous system for treating neuropsychiatric disorders associated with a dysfunction of the central dopaminergic system, such as Parkinson""s disease (Bonhomme et al., 1995), or for treating amnesic deficiencies as presented in patients suffering from Alzheimer""s disease (Marchetti-Gauthier et al., 1997). Such medicines might also be useful for treating peripheral disorders such as irritable bowel syndrome, gastroparesia, urinary incontinence and cardiac arrhythmias (Hedge and Eglen, 1996).
The receptors 5-HT4 present a unique pharmacology which is clearly different from that of the other members of the 5-HT receptor family (Ford and Clarke, 1993). Most pharmacological and transductional studies on the receptors 5-HT4 have been carried out on the central nervous system and gastro-intestinal tract of rodents and in porcine and human hearts (Eglen et al., 1995; Hedge and Eglen, 1996). Although the pharmacology of the receptors 5-HT4 present in these preparations is very similar, unexplained differences exist. Thus, benzamides, such as renzapride and cisapride, behave as potent and total agonists of the receptors 5-HT4 in mouse colliculi neurons, but are less potent and only partial agonists in the human heart (Ford and Clarke, 1993; Hoyer et al., 1994) and the detrusor muscle isolated from human bladder (Ford and Clarke, 1993, Hoyer et al., 1994; Candura et al., 1996). 5-Methoxytryptamine (5-MeOT), which is a 5-HT4 agonist, has an unusually weak agonist action on the receptors 5-HT4 of the human detrusor muscle (Candura et al., 1996). ML10302, an agonist of the receptor 5-HT4 which imitates the effect of 5-HT on the relaxation of rat oesophagus, and on electrically-induced contraction in guinea pig ileum (Langlois et al., 1994), has a weak agonist effect combined with a net antagonism of 5-HT-on the cAMP response generated by the human receptor 5-HT4(a) cloned from human atrium (Blondel et al., 1997), and an antagonist effect in colliculus neurons (Ansanay et al., 1996). In addition, the desensitization mechanisms for the receptors 5-HT4 are tissue-dependent. Specifically, a homologous rapid and total desensitization (cAMP-independent) of the receptors 5-HT4 is observed in mouse colliculi neurons (Ansanay et al., 1996) and rat oesophagus (Ronde et al., 1996), whereas this type of receptor is desensitized to a lesser degree in the human atrium (Kaumann et al., 1991).
The first receptor 5-HT4 was cloned from rat brain (Gerald et al., 1995), and two splicing variants (r5-HT4S and r5-HT4L) were identified. These variants differ in the lengths and the sequence of the carboxy-terminal end. The long form (r5-HT4L), which has also been cloned in mouse colliculi neurons (m5-HT4L), has transcripts in approximately each part of the brain (Claeysen et al., 1996). An interesting observation comes from the peripheral distribution of the transcripts of r5-HT4L and r5-HT4s in rats. Whereas both forms are expressed in the gastro-intestinal tract (ileum and colon), only the r5-HT4s transcript is found in the heart (Gerald et al., 1995). In addition, Sr-HT4s has been found exclusively in the atrium (Gerald et al., 1995). The human homologues of the receptors r5-HT4s and r5-HT4L, herein termed h5-HT4(a) and h5-HT4(b), have recently been cloned. The receptor h5-HT4(a) was cloned from human heart (Blondel et al., 1997; Claeysen et al., 1997); the receptor h5-HT4(b) was cloned from a library (Van de Wyngaert et al., 1997). These two receptors h5-HT4(a) and h5-HT4(b), when transiently expressed in COS-7 cells, present a conventional 5-HT4 pharmacological profile. However, the affinities of the cloned receptor h5-HT4(a) for agonists such as ML10302, BIMU1, renzapride and zacopride are lower than those found in the brain.
The inventors have now shown the existence of two novel serotonin receptor subtypes in humans, termed receptors h5-HT4(c) and h5-HT4(d).
The analysis of the sequence homologies suggests that forms h5-HT4(c) and h5-HT4(b) are respectively the human correspondents of forms r5-HT4s and r5-HT4L in rats, whereas forms h5-HT4(c) and h5-HT4(d) represent two novel isoforms of the receptor. The isoform h5-HT4(c) presents a high number of putative phosphorylation sites (one for protein kinase C, one for protein kinase A/protein kinase G and two for casein kinase II), all contained in the last 25 residues of the amino acid sequence.
The phosphorylation of the carboxy-terminal end of seven-transmembrane-domained receptors by protein kinase A regulates the desensitization of the receptor and the association of the receptor with G protein in response to increasing concentrations of cAMP (Dohlman et al., 1991).
In addition, in seven-transmembrane-domained receptors such as the xcex21- and xcex22-adrenergic receptors (Pei et al., 1994; Freedman et al., 1995) and the receptor 5-HT2c (Westphal et al., 1995), the phosphorylation of the receptor by non-cAMP-dependent protein kinases such as the xcex2-adrenergic receptor kinases (xcex2ARK1 and xcex2ARK2) or rhodopsin kinase allows substrate-activated homologous desensitization. The number and nature of the phosphorylation sites present at the C-terminal end of the receptor 5-HT4 splicing variants are thus capable of influencing the negative regulation of receptor function.
Tissue-dependent differences in receptor 5-HT4 desensitization mechanisms have been reported (Ford and Clarke, 1993), and may be related to the restricted profiles of the expression of the receptor 5-HT4 isoforms in the various tissues. Thus, the receptor 5-HT4 desensitization mechanism in mouse colliculi neurons resembles that described for xcex2-adrenergic receptors, and appears to be independent of the cAMP pathway. It has been proposed that a xe2x80x9cxcex2ARK-likexe2x80x9d kinase mediates the specific phosphorylation of the receptor 5-HT4 in these neuronal cells (Ford and Clarke, 1993). C-terminal phosphorylation of the isoform 5-HT4(c) by casein kinase II or PKC might thus explain the cAMP-independent desensitization observed in these cells.
The form 5-HT4(d) is characterized by a very short C-terminal end, with a coding sequence which finishes two amino acids after Leu358. Similarities in structure between the 5-HT4 C-terminal ends and the 5-HT7 C-terminal end (same number of variants, including a phosphorylation site-rich isoform and a truncated isoform close to the splicing site), despite the absence of a clear sequence homology, suggest similarities in the regulation of functional activity among the 5-HT receptors which are positively coupled to adenylate cyclase.
A subject of the present invention is an isolated polypeptide constituting splicing variants of the human serotoninergic receptor, the amino acid sequence of which is chosen from the sequence SEQ ID No. 2 of the variant polypeptide 5-HT4(c) and the sequence SEQ ID No. 4 of the variant polypeptide 5-HT4(d), and any biologically active polypeptide fragment or derivative of this polypeptide.
The present invention thus concerns more particularly an isolated polypeptide constituting the human serotoninergic receptor of type 5-HT4(c). More particularly, a subject of the invention is a polypeptide h5-HT4(c) having the amino acid sequence SEQ ID No. 2, or any biologically active polypeptide fragment or derivative of this polypeptide.
A subject of the present invention is also an isolated polypeptide constituting the human serotoninergic receptor of type 5-HT4(d). More particularly, a subject of the invention is a polypeptide h5-HT4(d) having the amino acid sequence SEQ ID No. 4, or any biologically active polypeptide fragment or derivative of this polypeptide.
The sequence SEQ ID No. 2 represents the amino acid sequence of the polypeptide h5-HT4(c) and the sequence SEQ ID No. 4 represents the amino acid sequence of the polypeptide h5-HT4(d).
xe2x80x9cDerivativexe2x80x9d is intended to mean any variant polypeptide of the polypeptide of sequence SEQ ID No. 2 or No. 4, or any molecule resulting from a modification of genetic and/or chemical nature of the sequence SEQ ID No. 2 or No. 4, i.e. obtained by mutation, deletion, addition, substitution and/or chemical modification of a single or of a limited number of amino acids, as well as any isoform sequence, i.e. a sequence which is identical to the sequence SEQ ID No. 2 or No. 4 or to one of its modified sequences or fragments, and which contains one or more amino acids in the form of a D enantiomer, said variant, modified or isoform sequences having conserved at least one of the properties which make them biologically active.
The expression xe2x80x9cbiologically activexe2x80x9d means that the compound to which it relates is capable of binding to serotonin or to serotonin-related ligands and/or of participating in serotonin-induced signal transduction at the cell membrane, in particular in adenylate cyclase activation, and/or is capable of inducing antibodies which recognize the polypeptide h5-HT4(c) or h5-HT4(d) according to the invention. Examples of serotonin-related ligands are in particular 5-methoxytryptamine, GR113808, BIMU1, etc.
The invention thus also comprises any polypeptide having an amino acid sequence which is essentially identical to the sequence SEQ ID No. 2 or No. 4, in which one or more residues have been constitutively substituted with a similar functional residue, and which demonstrates its suitability in mimicking the receptor h5-HT4(c) or h5-HT4(d) as described in the present invention. Examples of conservative substitutions include substituting a hydrophobic residue such as isoleucine, valine, leucine or methionine with another hydrophobic residue, substituting a polar hydrophilic residue such as arginine with lysine, glutamine with asparagine, or glycine with serine, substituting a basic residue such as lysine, arginine or histidine with another basic residue or substituting an acid residue such as aspartic acid or glutamic acid with another acid residue.
Similarly, the invention comprises any polypeptide having one or more residues which are chemically derived by reaction of a functional group. The polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions of residues with respect to the sequence SEQ ID No. 2 or No. 4, as long as the biological activity is maintained.
The polypeptides h5-HT4(c) and h5-HT4(d) of the present invention can be synthesized by all the methods well known to persons skilled in the art, including the recombinant DNA techniques. The polypeptides h5-HT4(c) or h5-HT4(d) can be synthesized by the techniques of synthetic chemistry, such as Merrifield-type synthesis, which is advantageous for reasons of purity, of antigenic specificity and of absence of undesired by-products, and for its ease of production.
A subject of the invention is also the isolated nucleotide sequence chosen from the sequence SEQ ID No. 1, the sequence SEQ ID No. 3, the nucleotide sequences derived from the sequence SEQ ID No. 1 or from the sequence SEQ ID No. 3 due to the degeneracy of the genetic code, to mutation, to deletion or to insertion, and the nucleotide sequences which are capable of specifically hybridizing with the sequence SEQ ID No. 1 or the sequence SEQ ID No. 3.
The sequence SEQ ID No. 1 represents the nucleotide sequence of the polypeptide h5-HT4(c) and the sequence SEQ ID No. 3 represents the nucleotide sequence of the polypeptide h5-HT4(d).
The various nucleotide sequences of the invention may or may not be of artificial origin. They can be DNA or RNA sequences which are obtained by screening sequence libraries by means of probes developed on the basis of the sequence SEQ ID No. 1 or No. 3. Such libraries can be prepared by conventional techniques of molecular biology which are known to persons skilled in the art.
The nucleotide sequences according to the invention can also be prepared by chemical synthesis or by mixed methods including the chemical or enzymatic modification of sequences obtained by screening libraries.
These nucleotide sequences allow the production of nucleotide probes which specifically hybridize with a sequence SEQ ID No. 1 or No. 3 according to the invention. The suitable hybridization conditions correspond to the conditions of temperature and of ionic strength usually used by persons skilled in the art, preferably to temperature conditions between (Tm minus 5xc2x0 C.) and (Tm minus 30xc2x0 C.), and more preferably to temperature conditions between (Tm minus 5xc2x0 C.) and (Tm minus 10xc2x0 C.) (high stringency), Tm being the theoretical melting temperature, which is defined as being the temperature at which 50% of the paired strands separate. Such probes also form part of the invention. They can be used as diagnostic tools in vitro for detecting, by hybridization experiments, in particular xe2x80x9cin situxe2x80x9d hybridization experiments, transcripts specific for the polypeptides of the invention in biological samples, or for detecting aberrant syntheses or genetic abnormalities resulting from a polymorphism, from mutations or from incorrect splicing.
The probes of the invention comprise a minimum of 10 nucleotides, and preferably at least 14 nucleotides, preferentially at least 20 nucleotides, more preferentially at least 50 nucleotides, and at the maximum comprise all of the nucleotide sequence SEQ ID No. 1 or SEQ ID No. 3 or of their complementary strands. In order to hybridize specifically with the sequences SEQ ID No. 1 or SEQ ID No. 3 encoding the receptors h5-HT4(c) or h5-HT4(d) respectively, and not with the nucleotide sequences encoding the receptors h5-HT4 (a) or h5-HT4(b), the probes according to the invention should contain a sequence specific for the 3xe2x80x2 end of the sequences SEQ ID No. 1 or No. 3 which encodes the C-terminal ends of the receptors h5-HT4(c) or h5-HT4(d).
Preferably, the probes of the invention are labelled prior to their use. For this, several techniques are easily accessible to persons skilled in the art, such as for example fluorescent, radioactive, chemiluminescent or enzymatic labelling.
The in vitro diagnostic methods in which these nucleotide probes are used for detecting aberrant syntheses or genetic abnormalities, such as loss of heterozygosis or gene rearrangement, in the nucleic acid sequences encoding a polypeptide h5-HT4(c) or h5-HT4(d) or a biologically active fragment are included in the present invention.
A subject of the invention is also a method for detecting the expression of the human receptor 5-HT4(c) or 5-HT4(d) in a cell or tissue sample, comprising the steps consisting in:
preparing the RNA of said sample;
bringing said RNA obtained into contact with a probe having a nucleotide sequence which is capable of specifically hybridizing with respectively the sequence SEQ ID No. 1 or the sequence SEQ ID No. 3 as defined above;
detecting the presence of mRNA which hybridizes with this probe, which indicates the expression of the receptor h5-HT4(c) or h5-HT4(d).
A subject of the invention is also a method for detecting the expression of the human receptor 5-HT4(c) or 5-HT4(d) in cells or a tissue by in situ hybridization, comprising the steps consisting in:
bringing said cells or said tissue into contact with a probe having a nicleotide sequence which is capable of specifically hybridizing with respectively the sequence SEQ ID No. 1 or the sequence SEQ ID No. 3 as defined above;
detecting the presence of mRNA which hybridizes with this probe, which indicates the expression of the receptor 5-HT4(c) or 5-HT4(d).
The cDNA probes of the invention can also advantageously be used for detecting chromosomal abnormalities.
The nucleotide sequences of the invention are also useful for the manufacture and the use of sense and/or antisense oligonucleotide primers for sequencing reactions or specific amplification reactions according to the so-called PCR (polymerase chain reaction) technique or any other variant of this technique.
The nucleotide sequences according to the invention have, moreover, uses in the therapeutic field, for preparing antisense sequences which are capable of specifically hybridizing with a nucleic acid sequence, including a messenger RNA, and which can be used in gene therapy. A subject of the invention is thus antisense sequences which are capable of at least partially inhibiting the production of polypeptides 5-HT4(c) or 5-HT4(d) as described above. Such sequences advantageously consist of those which constitute the reading frame encoding 5-HT4(c) or 5-HT4(d) in the transcript.
The nucleotide sequences according to the invention can, moreover, be used for producing recombinant polypeptides with a receptor 5-HT4(c) or 5-HT4(d), as defined above, activity.
These polypeptides can be produced from the nucleotide sequences defined above, according to techniques for producing recombinant products known to persons skilled in the art.
According to one embodiment of the invention, the nucleotide sequence can be inserted into an expression vector in which it is linked, in an effective manner, to elements which allow the regulation of its expression, such as in particular transcription terminators and/or promoters.
The signals which control the expression of the nucleotide sequences (promoters, activators, termination sequences, etc.) are chosen as a function of the host cell used. To this end, the nucleotide sequences according to the invention can be inserted into vectors which replicate autonomously in the chosen host or vectors which integrate into the chosen host. Such vectors will be prepared according to the methods currently used by persons skilled in the art, and the clones resulting therefrom can be introduced into a suitable host by standard methods, such as for example electroporation or calcium phosphate precipitation.
The cloning and/or expression vectors as described above, which contain one of the nucleotide sequences defined according to the invention, also form part of the present invention. Such an expression vector can be in particular a plasmid (such as pRc/CMV, which is available from Invitrogen, Carlsbad, Calif., or pUC18, which is available from Pharmacia, Piscataway, N.J.), a cosmid, a phage (such as the Lambda phage) or any type of recombinant virus.
The invention is also directed towards the host cells which are transiently or stably transfected by these expression vectors. These cells can be obtained by introducing into prokaryotic or eukaryotic host cells a nucleotide sequence which is inserted into a vector as defined above, and then culturing said cells under conditions which allow the replication and/or expression of the transfected nucleotide sequence.
Examples of host cells include, in particular, mammalian cells such as COS-7, CHO, NIH3T3, HeLa, LM(tkxe2x88x92), HEK293, etc. cells. The host cells can be in particular cell lines such as C6 glioma cells.
A subject of the invention is thus more particularly the cell lines which stably express the polypeptide 5-HT4(c) or 5-HT4(d) according to the invention.
Preferably, but not exclusively, the host cells for the expression of the functional recombinant receptor 5-HT4(c) or 5-HT4(d) express endogenous or recombinant G proteins and adenylate cyclases.
The host cells according to the invention can be used in a method for producing a polypeptide 5-HT4(c) or 5-HT4(d), this being a method in which cells transfected according to the invention are cultured under conditions which allow the expression of a polypeptide of sequence SEQ ID No. 2 of No. 4, or any biologically active fragment or derivative of this polypeptide, said biologically active polypeptide, fragment or derivative of this polypeptide is recovered, and then it is purified.
The purification methods used are known to persons skilled in the art. The recombinant polypeptide obtained can be purified from cell lysates and extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, the chromatography methods, the techniques of immunoaffinity using specific mono- or polyclonal antibodies, etc.
A subject of the invention is also poly- or monoclonal antibodies or fragments thereof, or chimeric or immunoconjugated antibodies. These antibodies or fragments are characterized in that they are obtained from a receptor polypeptide as defined above, derivative or polypeptide fragment of this polypeptide, which is administered to an animal, and are capable of specifically recognizing a polypeptide 5-HT4(c) or 5-HT4(d).
Polyclonal antibodies can be obtained from the serum of an animal immunized against the receptor 5-HT4(c) or 5-HT4(d) according to the usual procedures.
According to one embodiment of the invention, as an antigen, a suitable peptide fragment can be used which can be coupled, via a reactive residue, to a protein or another peptide bearing a T-dependent epitope. Rabbits are immunized with the equivalent of 1 mg of the peptide antigen according to the procedure described by Benoit et al (1982). At four-week intervals, the animals are treated with injections of 200 xcexcg of antigen, and bled 10 to 14 days later. After the third injection, the antiserum is examined so as to determine its capacity for binding to the iodine-radiolabelled peptide antigen, which is prepared by the chloramine-T method, and is then purified by chromatography on a carboxymethylcellulose (CMC) ion exchange column. The antibody molecules are then collected from the mammals and isolated to the desired concentration by the methods which are well known to persons skilled in the art, for example using DEAE Sephadex to obtain the IgG fraction.
Another protocol for obtaining polyclonal antibodies is described in Example 8.
To increase the specificity of the polyclonal serum, the antibodies can be purified by immunoaffinity chromatography using solid phase-immunizing polypeptides. The antibody is brought into contact with the solid phase-immunizing polypeptide for a sufficient length of time, so as to immunoreact the polypeptide with the antibody molecule to form a solid phase immunological complex.
The monoclonal antibodies can be obtained according to the conventional method of hybridoma culture described by Kxc3x6hler and Milstein (1975).
The antibodies or antibody fragments of the invention are for example chimeric antibodies, humanized antibodies, or Fab and F(abxe2x80x2)2 fragments. They can also be in the form of immunoconjugates or of labelled antibodies. For example, they can be combined with a toxin, such as the diphtheria toxin, or a radioactive product. In this case, these immunotoxins may constitute therapeutic agents which can be used for treating certain pathologies involving an overexpression of the receptor 5-HT4(c) or 5-HT4(d).
The antibodies of the invention, in particular the monoclonal antibodies, can also be used for the immunohistochemical analysis of the receptors 5-HT4(c) or 5-HT4(d) on specific- tissue sections, for example by immunofluorescence, gold labelling, immunoperoxidase, etc.
The anti-5-HT4(c) or anti-5-HT4(d) antibodies can be advantageously used in any situation where the expression of the receptor 5-HT4(c) or 5-HT4(d) must be observed (abnormal overexpression, monitoring the regulation of the membrane expression, etc.).
The invention also concerns a method for diagnosing in vitro an abnormal accumulation or expression of receptor 5-HT4(c) or 5-HT4(d) in a biological sample, and/or for measuring the expression level of this receptor in said sample, comprising bringing at least one antibody as defined above into contact with said biological sample under conditions which allow the possible formation of specific immunological complexes between a receptor 5-HT4(c) or 5-HT4(d) and said antibody(ies) and detecting the specific immunological complexes possibly formed.
A subject of the invention is also a kit for diagnosing in vitro an abnormal expression of the receptor 5-HT4(c) or 5-HT4(d) in a biological sample and/or for measuring the expression level of the receptor 5-HT4(c) or 5-HT4(d) in said sample comprising:
at least one antibody specific for the receptor 5-HT4(c) or 5-HT4(d), optionally bound to a support;
means for revealing the formation of specific antigen/antibody complexes between the receptor 5-HT4(c) or 5-HT4(d) and said antibody, and/or means for quantifying these complexes.
A subject of the invention is also the use of the polypeptide 5-HT4(c) or 5-HT4(d) according to the invention for detecting the presence of anti-receptor autoantibodies in pathologies associated with the dysfunction of the receptors.
A subject of the invention is also a pharmaceutical composition comprising a receptor 5-HT4(c) or 5-HT4(d) polypeptide, derivative or polypeptide fragment as defined above, a nucleotide sequence as defined above, or an antibody as defined above, combined with a pharmaceutically acceptable vehicle.
A subject of the invention is in particular a pharmaceutical composition which contains an antibody directed against the receptor 5-HT4(c) or 5-HT4(d), in sufficient amount to block the binding of the natural substrates to the receptor 5-HT4(c) or 5-HT4(d), and a pharmaceutically acceptable vehicle.
A pharmaceutical composition according to the invention in particular can be administered orally, parenterally, intravenously, intramuscularly, subcutaneously, percutaneously, or by intranasal administration.
The preparation of pharmaceutical compositions which contain active principles dissolved or dispersed in these latter is well known to persons skilled in the art. Generally, these compositions are prepared in the form of injectable solutions or suspensions. However, they can also be in solid forms which are suitable for extemporaneously preparing solutions or suspensions. The preparations can also be emulsified.
The methods of administration, the doses and the galenic forms of the pharmaceutical compositions according to the invention can be determined according to the criteria which are generally taken into account for establishing a therapeutic treatment suited to a patient, such as for example the age or the bodyweight of the patient, the seriousness of his/her general state, the tolerance to the treatment and the observed side effects, etc.
A subject of the invention is also a therapeutic treatment method for an individual suffering from complaints or disorders which are lessened, or even eliminated, by reducing the expression of the receptors 5-HT4(c) or 5-HT4(d), said method comprising administering to said individual an effective amount of the pharmaceutical composition as described above. The binding of the antibody to the receptor 5-HT4(c) or 5-HT4(d) in fact prevents the activation of the receptor and thus neutralizes the effects of an abnormal overexpression of these receptors.
A subject of the invention is also a method for screening compounds which are capable of binding to the polypeptide 5-HT4(c) or 5-HT4(d) according to the invention, in which said compounds are brought into contact with said polypeptide 5-HT4(c) or 5-HT4(d), and the degree of binding between said compounds and said polypeptide 5-HT4(c) or 5-HT4(d) is evaluated.
A large number of compounds can thus be rapidly screened to test the capacity of these compounds for binding to the receptor 5-HT4(c) or 5-HT4(d) according to the invention.
These binding assays can also be used for determining the presence or absence of serotonin in a biological sample, as well as for isolating novel endogenous ligands.
A subject of the invention is thus also a method for identifying ligands of the receptor 5-HT4(c) or 5-HT4(d) comprising the steps consisting in:
a) bringing a biological sample which is likely to contain ligands of the receptor into contact with a polypeptide 5-HT4(c) or 5-HT4(d) or a host cell expressing said polypeptide,
b) isolating the ligand-receptor complexes formed,
c) identifying the ligands of the receptor 5-HT4(c) or 5-HT4(d).
A subject of the invention is also a method for diagnosing in vitro an abnormal accumulation or expression of serotonin or analogues thereof in a biological sample, and/or for measuring its (their) expression level in said sample, comprising bringing a polypeptide according to the invention into contact with said biological sample under conditions which allow the formation of specific immunological complexes between said polypeptide and serotonin or analogues thereof, and detecting the specific immunological complexes formed.
The binding assays used in the context of the present invention can be carried out according to methods well known to persons skilled in the art. In particular, the compounds which are capable of binding to the receptor polypeptide can be prelabelled, and can be used alone or in competition with other unlabelled compounds.
More particularly, competitive binding assays can for example be carried out, i.e. assays of ELISA or IRMA type.
In the context of the invention, labelling means can be used for detecting the receptor 5-HT4(c) or 5-HT4(d) polypeptide according to the invention, the anti-5-HT4(c) or anti-5-HT4(d) antibodies and/or the ligands of the receptor 5-HT4(c) or 5-HT4(d).
The labelling means used can be in particular a fluorescent labelling agent which binds chemically to antibodies or to antigens without denaturation, so as to form a colorant fluorochrome which is a useful immunofluorescent indicator. The labelling agent can also be an enzyme, such as peroxidase (HRP) or glucose oxidase. Radioactive elements can also be used as labelling agents.
The inventors have moreover studied the tissue distribution of the four various splicing variants of the receptor 5-HT4, i.e. 5-HT4(a), 5-HT4(b), 5-HT4(c) and 5-HT4(d).
The expression profile of each splice variant of 5-HT4 is not restricted to a given tissue, with the exception of 5-HT4(d), which is only in the intestines. Some tissues (human atrium, brain, intestine) express three or four 5-HT4 isoforms, whereas others (bladder, kidney) express only one isoform. Thus, the bladder specifically expresses only the 5-HT4(a) form. In two tissues (ventricles of the heart and liver) no transcript encoding any 5-HT4 splicing variant could be amplified. The presence of the receptor 5-HT4(d) exclusively in the intestines makes a potential therapeutic target thereof for treating digestive disorders associated with this receptor, without side effects on the organs expressing the other isoforms. The cerebral location of the 5-HT4(c) isoform, although not exclusive, makes it possible to envisage that this isoform participates in the neuronal effects of serotonin. Specifically, it has been shown that the receptors 5-HT4 of mouse colliculi neurons, unlike the receptors 5-HT4 of the human atrium (Kaumann et al., 1991), are rapidly desensitized in the presence of serotonin (Ansanay et al., 1996). Now, the h5-HT4(c) isoform is the only one to possess phosphorylation sites on its C-terminal end, making it more sensitive to homologous desensitization.
A subject of the invention is thus more particularly the use of a receptor 5-HT4(c) polypeptide according to the invention for screening molecules which are useful for manufacturing medicines intended for treating the disorders of the central nervous system associated with the abnormal expression of the receptor 5-HT4(c).
A subject of the invention is also the use of a receptor 5-HT4(d) polypeptide according to the invention for screening molecules which are useful for manufacturing medicines intended for treating the disorders of the gastro-intestinal tract associated with the abnormal expression of the receptor 5-HT4(d).
The inventors have also studied the pharmacological profile of the receptors 5-HT4(c) or 5-HT4(d) according to the invention. Whereas the 5-HT4(a), 5-HT4(b) and 5-HT4(d) subtypes expressed in COS-7 cells present a similar capacity for adenylate cyclase coupling when they are exposed to 5-HT, the expression of the 5-HT4(c) isoform gives a constitutive activation of the adenylate cyclase, which results in an increase in the basal level of cAMP. The degree of constitutive coupling is also increased by overexpressing the 5-HT4(c) isoform, by using more plasmid DNA for the transcription. Such a constitutive coupling has been described in several other situations, for example 1) in the metabotropic glutamate receptor family, some splicing variants being spontaneously coupled to G proteins (Prezeau et al., 1996); 2) in specific experimental or pathological mutations which lead to a constitutive activity in certain receptors (Coughlin, 1994); 3) in the overexpress on of 7-transmembrane-domained receptors (Kenakin, 1996) such as the receptor m5-HT4L (Claeysen et al., 1996), which leads to a constitutive activation of Gs.
All the receptor h5-HT4 subtypes expressed in the COS-7 cells present a conventional receptor 5-HT4 profile in terms of order of potency of diverse serotoninergic ligands tested (Hoyer et al., 1994). No major difference could be found between the isoforms (a), (b) and (c) as regards the affinity constants of the 5-HT4 agonists and antagonists. However, the isoform (d) always presents a higher. affinity for any 5-HT4 ligand tested, in comparison with the other isoforms (Table 1). Since the receptor 5-HT4(d) is the shortest of the four isoforms of the receptor, this result suggests that the modifications of the C-terminal protein sequence of the 5-HT4 receptors can induce changes in the binding properties. The question is to know whether these modifications participate in the variability of the affinity constants for the various 5-HT4 ligands observed in the various tissues of animal species (Blondel et al., 1997). Alternative mechanisms for these variabilities may include species differences for the isoforms of the receptor 5-HT4 or the existence of internal splicing variants (Ullmer et al., 1995) which might more directly affect the ligand binding site.
A subject of the invention is also a method for evaluating the pharmacological properties of the compounds which are capable of binding to the receptor 5-HT4(c) or 5-HT4(d) polypeptide, termed receptor 5-HT4(c) or 5-HT4(d) ligands, comprising the steps consisting in:
a) culturing cells which express the polypeptide 5-HT4(c) and/or 5-HT4(d) in the presence of at least one receptor 5-HT4(c) and/or 5-HT4(d) ligand; and
b) evaluating the capacity of the ligand for modifying signal transduction.
More particularly, the capacity of a ligand for modifying signal transduction can be evaluated by determining the concentration of intracellular cyclic AMP (cAMP) formed by the activation of adenylate cyclase, or the adenylate cyclase activity.
According to one variant of this embodiment, said cells can be advantageously cultured in the presence:
either of increasing concentrations of at least one receptor 5-HT4(c) or 5-HT4(d) ligand, the capacity for modulating signal transduction of which is desired and of a given concentration of at least one known agonist of 5-HT4(c) or 5-HT4(d);
or of increasing concentrations of at least one known agonist of 5-HT4(c) or 5-HT4(d) and of a given concentration of at least one receptor 5-HT4(c) or 5-HT4(d) ligand, the capacity for modulating signal transduction of which is desired.
The capacity of said ligand for modulating signal transduction is then evaluated by quantitatively determining the expression of said reporter gene, as a function of the concentration of said ligand.
The biological assays according to the invention as described above thus make it possible to evaluate the pharmacological profile of the tested compounds, in particular to determine whether the tested compounds are capable of acting as agonists or antagonists of the receptor 5-HT4(c) or 5-HT4(d) according to the invention.
The present inventors have thus shown that, surprisingly, ML10375, while antagonizing the 5-HT-induced cAMP response mediated by all the 5-HT4 isoforms, reduces basal adenylate cyclase activity only in the cells transfected with the receptors 5-HT4(c) and 5-HT4(d). This phenomenon is mediated by a reverse agonist effect of ML10375. This phenomenon represents the first of its kind in the family of receptors 5-HT4, and it suggests that the C-terminal end structure of the 7TM receptor may participate in the development of a reverse agonist effect of a given antagonist. The compound ML10375 of formula 2-(cis-3,5-dimethyl-piperidino)ethyl 4-amino-5-chloro-2-methoxybenzoate is particularly useful for preparing medicines with reverse agonist action towards the receptor 5-HT4(c) or 5-HT4(d). This reverse agonist property is different from the receptor 5-HT4 selective antagonist property already known for the compound ML10375 (patent application EP 683 161). Specifically, a simple antagonist acts only by competition with the natural ligand (serotonin), by preventing the action of the latter. As for the reverse agonist, it can lower a receptor activity which is too high in the absence of natural ligand. Thus, in the case of a pathological overexpression of receptors 5-HT4(c) or 5-HT4(d) in the brain (for 5-HT4(c)), the heart (for 5-HT4(c)) or the digestive system (for 5-HT4(c) and 5-HT4(d)), ML10375 can normalize the response by reducing the intrinsic activity of the receptors. Such an effect cannot be obtained with another molecule which would act as a simple selective antagonist of these receptors.
The examples and figures, the legends of which are below, illustrate the invention without limiting it:
FIG. 1A represents the deduced amino acid sequences of the C-terminal end of the splicing variants of receptor 5-HT4 in rats and humans. In both species, the sequence diverges after Leu358. The amino acid differences between rats and humans are high-lighted. The white circles correspond to the protein kinase consensus site, the black circles to the casein kinase II consensus site and the black triangle to a protein kinase A/protein kinase G consensus site. The asterisk corresponds to the terminal stop codon.
FIG. 1B represents the compared amino acid sequences of the variants 5-HT4(c) and 5-HT4(d).
The seven potential transmembrane domains (TM1 to TM7) of the proteins are underlined, and the putative phosphorylation sites are indicated with suitable symbols.
FIG. 2 represents the RT-PCR analysis carried out with 50 ng of mRNA from diverse human tissues. The PCR products were separated on a 1.5% agarose gel and analysed by Southern Blot using a specific internal oligonucleotide probe labelled with 32P, this being a primer common to 5-HT4(a), 5-HT4(b), 5-HT4(c) and 5-HT4(d). An 8-hour exposure of the autoradiograph is presented. A positive control was carried out using rat actin primers on mRNA samples treated with (+RT) or without (xe2x88x92RT) reverse transcriptase. The PCR products of the control experiment were analysed on a 1.5% agarose gel, and a photograph of the ethidium bromide-stained gel is presented. The PCR primers used for this analysis are described in Examples 1 and 2. (A: atrium, V: ventricle, Br: brain, G: gastro-intestinal tract, L: lung, K: kidney, Bl: bladder).
FIG. 3 represents the [3H]GR113808 saturation binding analysis on membrane preparations of COS-7 cells expressing isoforms 5-HT4(a) (A), 5-HT4(b) (B), 5-HT4(c) (C) and 5-HT4(d) (D). The membranes collected from the transiently transfected COS-7 cells were incubated with 8 concentrations of [3H]GR113808 (0.02-3.5 nM) for 30 minutes at. 25xc2x0 C. The nonspecific binding was defined with 10 xcexcM of ML10375. The results are from a single experiment, but are representative of three identical experiments. The Kd and Bmax values are determined by computer-assisted nonlinear regression analysis (GraphPad, Prism software).
FIG. 4 represents the inhibition, by 5-HT of the specific binding of [3H]GR113808 to the cloned receptors 5-HT4(a) (FIG. 4A), 5-HT4(b) (FIG. 4B), 5-HT4(c) (FIG. 4C) and 5-HT4(d) (FIG. 4D). The membranes of the transiently transfected COS-7 cells were incubated with a concentration of [3H]GR113808 which was equal to 50% of the Kd value of each isoform of the receptor. The nonspecific binding was defined with 10 xcexcM ML10375. The results are presented as a percentage of specific binding in the absence of 5-HT; the data were analysed by computer-assisted nonlinear regression analysis (GraphPad, Prism software).
FIG. 5 represents the cAMP responses to diverse agonists and antagonists of the receptor 5-HT4 using the receptors 5-HT4(a), 5-HT4(b), 5-HT4(c) and 5-HT4(d) transiently expressed in the COS-7 cells, or in the xe2x80x9cblankxe2x80x9d-transfected cells. The cells were preincubated with 5 mM theophylline and 10 xcexcM pargyline for 15 minutes, and then incubated with 1 xcexcM agonists (5-HT: A, C, D and E; ML10302: B and C; renzapride: B and D) and/or antagonists (ML10375: B and E), or 10 xcexcM forskolin (A), for 15 minutes. The effect of the agonist or of the antagonist of 5-HT-induced cAMP accumulation was tested by adding the agonist or the antagonist for the 15-minute preincubation period, followed by adding 5-HT for 15 minutes. The values are mean valuesxc2x1SEM of 7 to 12 experiments. NS: not significant: *, p less than 0.05 and **, p less than 0.01.
FIG. 6 represents the cAMP responses of 5-HT (1 xcexcM), of forskolin (10 xcexcM) and of 5-HT4 antagonist ML10375 (1 xcexcM), using the receptor 5-HT4(c) transiently expressed in the COS-7 cells, or in the xe2x80x9cblankxe2x80x9d-transfected cells as control. The cells are transfected using 4 to 8 xcexcg per well (A and B) or 6 xcexcg per well (C and D) of plasmid DNA. The incubation conditions are identical to those described in FIGS. 5C and D: the responses are studied in the presence or absence of a 16-hour preincubation with PTX (100 ng/ml). The values are mean valuesxc2x1SEM of 6 to 11 experiments. NS: not significant; *, p less than 0.05 and **, p less than 0.01.
FIG. 7 represents the saturation analysis of the [3H]GR113808 binding on membrane preparations of CHO cells expressing isoforms 5-HT4(c) (A) and 5-HT4(d) (B). The membranes collected from the stably transfected CHO cells were incubated with 8 concentrations of [3H]GR113808 (0.01-2.5 xcexcM) for 30 minutes at 25xc2x0 C. The nonspecific binding was defined with 10 xcexcM of ML10375. The results are from a single experiment, but are representative of three identical experiments. The Kd and Bmax values (FIG. 7C and 7D) are determined by computer-assisted nonlinear regression analysis (GraphPad, Prism software).
FIG. 8 represents the cAMP responses to serotonin using the receptors 5-HT4(c) and 5-HT4(d) stably expressed in the CHO cells, or in the xe2x80x9cblankxe2x80x9d-transfected cells. The cells were preincubated with 5 mM theophylline and 10 xcexcM pargyline for 15 minutes, and then incubated with 1 xcexcM 5-HT. The results are from a single experiment, but are representative of three identical experiments.
FIG. 9 represents an ELISA assay on sera from rabbits immunized with the peptides corresponding to various sequences derived from the isoforms of the receptor 5-HT4. The sequences of these peptides are given in Example 8.
FIG. 9 gives the optical density values of an immunoenzymatic assay on the respective peptides adsorbed (5 xcexcg/ml in a carbonate buffer at pH=9.5) onto MAXIsorb plates (Nunc, Denmark). The antisera were incubated for one hour at 37xc2x0 C., and revealed with a peroxidase-coupled goat anti-rabbit IgG antibody conjugate (dilution {fraction (1/10,000)}) and the substrate H2O2-ABTS (calorimetric indicator of oxidoreduction for peroxidase).
FIG. 10 demonstrates, by Western Blot, the presence of the receptor 5-HT4 in the CHO cells stably expressing the 5-HT4(a) (line 2) and 5-HT4(c) (line 3) isoforms. The CHO cells were transfected with the expression vectors encoding the forms (a) and (c) of the receptor 5-HT4 and selected for their neomycin resistance. Fifty xcexcg of proteins originating from membrane extracts are separated on a 10% polyacrylamide gel, and then transferred onto nitrocellulose membrane. After incubation for 16 h in the presence of 60 xcexcg of anti-5-HT4 antibody (G21V), the blot is revealed by chemiluminescence (ECL, Amersham) and scanned. Line 1 indicates the result obtained on the control CHO cells, in which no labelling is detected; lines 2 and 3 originate from clones of CHO cells overexpressing respectively the receptor 5-HT4(a) and 5-HT4(c). A band migrating approximately with the size of 60 kDa is visualized.
FIG. 11 demonstrates, by Western Blot, the presence of the receptors h5-HT4 in the CHO cells stably expressing the h5-HT4(c) (FIG. 11a) and 5-HT4(d) (FIG. 11b) isoforms. The CHO cells were transfected with the expression vectors encoding the forms (c) and (d) of the receptor h5-HT4 and selected for their neomycin resistance. Sixteen xcexcg of protein coming from CHO cells are separated on a 10% polyacrylamide gel, and then transferred onto cellulose membrane. After 16 h of incubation in the presence of 16 xcexcg of anti-h5-HT4(c) (anti-C21S, FIG. 11a) or anti-5-HT4(d) (anti-C7F, FIG. 11b) antibodies, and in the presence (2nd line) or absence (1st line) of 50 xcexcg of the corresponding peptides, the blots are revealed by chemiluminescence (ECL, Amersham) and scanned. The second line indicates the bands of the receptor which are nonspecific, since they are not inhibited by the corresponding peptides. The anti-C21S antibody recognizes two specific bands at 44 and 60 kDa. The anti-C7F antibody recognizes a specific band at 40 kDa. The band at 60 kDa corresponds to the glycosylated receptor and the bands at 44 and 40 kDa to the nonglycosylated receptor.


Federico Pedrocchi

Federico Pedrocchi (1 May 1907 – 20 January 1945) was an Italian comic book artist and writer. He sometimes used the pen name Costanzo Federici. He founded the magazine Paperino e altre avventure (Donald Duck and Other Adventures) in 1937, and wrote some of the earliest Disney comic book stories.

Life and career
Pedrocchi was born in Buenos Aires, Argentina of Italian parents, and he moved back to Italy with his family in 1912. Forced to interrupt his studies because of his father's death, in 1930 he opened a small advertising studio and the same year he started collaborating as illustrator with the children magazine  Corriere dei Piccoli and with a number of other magazines, notably La Domenica del Corriere. In 1935 he debuted as a cartoonist with the story  "I due tamburini", and in 1936 he started collaborating with Cesare Zavattini for the script of Saturno contro la Terra.

In 1937 Pedrocchi was allowed by  Disney to write and draw stories with Disney characters, and the same year he founded the magazine Paperino e altre avventure (Donald Duck and Other Adventures), published by Mondadori, in which Donald Duck became the protagonist of long, adventurous stories. Pedrocchi wrote eight serials starring Disney characters for the paper, featuring Donald Duck, Goofy (Pippo) and the characters from Snow White and the Seven Dwarfs (Biancaneve e i Setti Nani). During the same period, he also wrote two further Disney stories which were printed in Mondadori's biweekly publication Albi d'oro.

Following the success of the magazine, he was appointed editorial and artistic director of Mondadori's  comic magazines in 1939, a role he maintained until he was enlisted in the army in 1942. Pedrocchi died at 37 years old, after having been hit by a burst of machine gun during an English air raid.

Disney comics bibliography
Pedrocchi's Disney stories in Paperino e altre avventure were:

 Paolino Paperino e il mistero di Marte (Donald Duck and the Secret of Mars): issues #1-18
 Paolino Paperino inviato speciale (Donald Duck, Special Correspondent): #19-48
 Pippo viaggiatore di commercio (Goofy, Business Traveler): #40-48
 Paperino fra i pellirosse (Donald Duck Among the Redskins): #72-100
 Biancaneve e il mago Basilisco (Snow White and the Basilisk Wizard): #72-100
 Paperino chiromante (Donald Duck, Fortune Teller): #101-130
 I Sette Nani cattivi contro i Sette Nani buoni (The Seven Bad Dwarves Against the Seven Good Dwarfs): #101-121
 Paperino e il vaso cinese (Donald Duck and the Chinese Vase): #131-149

The stories published in Albi d'oro were:
 Paperino e la pietra filosofale (Donald Duck and the Philosopher's Stone): #22/1938
 Clarabella fra gli artigli del diavolo nero (Clarabelle in the Claws of the Black Devil): #25/1939

References

Category:Italian comics artists
Category:Italian comics writers
Category:1907 births
Category:1945 deaths
Category:People from Buenos Aires
Category:Disney comics writers
Category:Disney comics artists


Results from a prospective acute inpatient rehabilitation database: clinical characteristics and functional outcomes using the Functional Independence Measure.
Rehabilitation improves functional outcomes, but there is little data on the profiles and outcomes of patients undergoing inpatient rehabilitation in Singapore. The aims of this paper were to document the clinical characteristics and functional outcomes, using the Functional Independence Measure (FIM), of all patients admitted to an inpatient rehabilitation unit in a tertiary teaching hospital, and to identify and analyse factors significantly associated with better discharge functional scores and higher functional gains. In this prospective cohort study over a 4-year period, clinical and functional data for 1502 patients admitted consecutively to the Singapore General Hospital inpatient rehabilitation unit were charted into a custom-designed rehabilitation database. The primary outcome measures were the discharge total FIM scores, FIM gain and FIM efficiency. Multiple linear regression analysis was used to identify independent variables associated with better discharge FIM scores and FIM gain. The mean age was 61.3 +/- 15.0 years and 57.2% of the patients were male. Stroke (57.9%) followed by spinal cord injury (9.7%) were the most common diagnoses. The average rehabilitation length of stay was 21.5 +/- 19.0 days. The mean admission total FIM score was 70.3 +/- 23.2 and the mean discharge total FIM score was 87.3 +/- 23.0, with this gain being highly significant (P <0.001). The mean FIM gain was 17.0 +/- 13.4 and FIM efficiency was 0.95 +/- 0.90 points/day. Factors associated with better functional outcomes were higher admission motor and cognitive FIM scores, male gender, a longer rehabilitation length of stay and the use of acupuncture. Factors associated with poorer functional outcomes were older age, clinical deconditioning, ischaemic heart disease, depression, pressure sores and the presence of a domestic worker as a caregiver. The FIM is an easy-to-use, standardised and robust general measure of functional disability. Multiple demographic, clinical and socio-cultural variables are associated with the primary functional outcomes and should be taken into account in rehabilitation and discharge planning. Nevertheless, rehabilitation improves functional outcomes across a wide range of diagnoses. Further research should be aimed at evaluating long-term disability postdischarge from inpatient rehabilitation and translating these findings into improving rehabilitation and healthcare resource utilisation.


Disappointing. Dismal. Bleak. These are just a few of the words used to describe the latest employment report. It showed that the U.S. economy added just 69,000 jobs in May. That's less than half of what economists had expected. And this was the third consecutive month of weak results, which raises new concerns about the sputtering economic recovery.

So with the unemployment rate now at 8.2 percent, we asked people in Athens, Ohio and in Washington, D.C., how they're faring in this economy.

AMY YANNITY: My name is Amy Yannity and I work at the college bookstore, getting by like most people. Nothing great, you know. Nothing elaborate. But I came in before the computer and everything, so all the other kids are way ahead of me.

MARISOL ALCALDE: My name is Marisol Alcalde and I'm a college student at Trinity Washington University. It's definitely tough. You know, like I've looked around for jobs and none of them are hiring. And it seems like if they are they never hit you back up. But I'm expecting good change 'cause I'm just hopeful person. I'm just a very hopeful person. You got to keep looking. You never know.

(LAUGHTER)

CATHERINE BALL: Am I hopeful? Not really. I feel like it's going to get even worse. My name is Catherine Ball. I've been working at Potbelly Sandwich Shop for four and a half months. I get - I was here but my money's gone so quick. So it's just hard making minimum wage, I guess. It's good to have a job though. You know, I can't complain. I'm a hard worker, always been a hard worker.

GARY GINTHER: My name is Gary Ginther and I'm a fine arts librarian for Ohio University Libraries, making the necessary cutbacks here and there. We're really cut very thin right now. I think we probably discovered how much we could tighten the belt for the first time in a long time. I think it's a cyclical thing, the economy. And so, I know that it's going to ebb and flow. You know, I'm old enough now that I've experienced this more than once.


This one-day conference will bring together researchers from across Canada who have examined thousands of writing assignments and interviewed dozens of faculty members in an effort to understand the role of writing in the sciences, engineering, mathematics, education, and the arts.


Sensitivity to register selection errors amongst 5- and 7-year-old children.
We are expected to behave appropriately to suit social situations. One form of behavioral control is the selection of a linguistic register that is appropriate to the listener. Register selection errors can sometimes be interpreted as rude behavior and result in having a bad influence on the relationship with the listener and the evaluation by other people. Previous studies have demonstrated that children begin to show sensitivity to registers from about 2 years of age, and that 5- to 6-year-old children understand the appropriate relationships between a listener and a register. However, it remains unclear whether and when children understand the influence made by register selection errors on listeners, and how children evaluate speakers when they make errors in register selection. Therefore, we investigated the above issues in Japanese children, who are required to use registers strictly. Also, we conducted a control study with Japanese-speaking adults to identify adults' evaluation of register selection errors. The results indicated that 7-year-old, but not 5-year-old children understood that the selection of an appropriate register gives listeners positive feelings. Moreover, 7-year-old children used register selection as an index of selective learning and social preference, although how they use others' register use skills for evaluating them was slightly different from adults. These results suggest that although children notice the social meanings behind the use of registers by the age of 7, a more extended period of development is required to master the social meaning of register selection. (PsycINFO Database Record (c) 2019 APA, all rights reserved).


Q:

Can I join a game in the middle of a 1st playthrough if I'm just starting a 2nd playthrough?

Some of my friends are playing Dead Island, but they are hopelessly behind me in terms of story progression. Will I be able to still join their game if I'm in an earlier chapter of a 2nd playthrough and they are in a later chapter of a 1st playthrough?
To put it simply, can I join a 1st playthrough chapter 5 game if my game is 2nd playthrough chapter 2?

A:

You can't find a game in a chapter you haven't reached in your current playthrough, so starting a 2nd playthrough will lock you out of public games in later chapters. However, you can be invited to a game with any level of progress, and will get a prompt saying quest progress will not be recorded if you are in an earlier chapter than the game you are trying to join.