Autistic traits in a sample of adult patients with schizophrenia: prevalence and correlates.

BACKGROUND: Schizophrenia and autism spectrum disorder (ASD) are currently conceptualized as distinct disorders. However, the relationship between these two disorders has been revisited in recent years due to evidence that they share phenotypic and genotypic expressions. This study aimed to identify ASD traits in patients with schizophrenia, and to define their demographic, psychopathological, cognitive and functional correlates. METHOD: Seventy-five schizophrenia patients (20 females, mean age 42 ± 12) were evaluated with the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview-Revised (ADI-R). Participants were also assessed with clinical, neuropsychological, and psychosocial functioning measures. RESULTS: Of the 75 patients, 47 were negative to all the autism scales administered (ADOS-TOT-NEG), 21 patients were positive to the ADOS Language sub-domain (ADOS-L-POS), 21 patients were positive to the ADOS Reciprocal Social Interaction (RSI) sub-domain (ADOS-RSI-POS), 14 patients were positive to the ADOS Total scale (ADOS-TOT-POS), and nine patients were positive to the ADI-R scale (ADI-R-POS). Demographic (duration of illness), psychopathological (negative symptoms and general psychopathology), and cognitive (working memory and processing speed) differences emerged between schizophrenic patients with and without ASD traits, while no differences in psychosocial functioning were detected. Results of this study indicate the existence, in a sample of patients with a diagnosis of schizophrenia, of a distinct group of subjects with ASD features, characterized by specific symptomatological and cognitive profile. CONCLUSIONS: These findings may contribute to better characterize patients with schizophrenia in order to develop new procedures and therapeutic tools in a more personalized perspective.

Adult Autism Subthreshold Spectrum (AdAS Spectrum): Validation of a questionnaire investigating subthreshold autism spectrum.

AIM: Increasing literature has shown the usefulness of a dimensional approach to autism. The present study aimed to determine the psychometric properties of the Adult Autism Subthreshold Spectrum (AdAS Spectrum), a new questionnaire specifically tailored to assess subthreshold forms of autism spectrum disorder (ASD) in adulthood. METHODS: 102 adults endorsing at least one DSM-5 symptom criterion for ASD (ASDc), 143 adults diagnosed with a feeding and eating disorder (FED), and 160 subjects with no mental disorders (CTL), were recruited from 7 Italian University Departments of Psychiatry and administered the following: SCID-5, Autism-Spectrum Quotient (AQ), Ritvo Autism and Asperger Diagnostic Scale 14-item version (RAADS-14), and AdAS Spectrum. RESULTS: The AdAS Spectrum demonstrated excellent internal consistency for the total score (Kuder-Richardson's coefficient=.964) as well as for five out of seven domains (all coefficients>.80) and sound test-retest reliability (ICC=.976). The total and domain AdAS Spectrum scores showed a moderate to strong (>.50) positive correlation with one another and with the AQ and RAADS-14 total scores. ASDc subjects reported significantly higher AdAS Spectrum total scores than both FED (p<.001) and CTL (p<.001), and significantly higher scores on the Childhood/adolescence, Verbal communication, Empathy, Inflexibility and adherence to routine, and Restricted interests and rumination domains (all p<.001) than FED, while on all domains compared to CTL. CTL displayed significantly lower total and domain scores than FED (all p<.001). A significant effect of gender emerged for the Hyper- and hyporeactivity to sensory input domain, with women showing higher scores than men (p=.003). A Diagnosis* Gender interaction was also found for the Verbal communication (p=.019) and Empathy (p=.023) domains. When splitting the ASDc in subjects with one symptom criterion (ASD1) and those with a ASD, and the FED in subjects with no ASD symptom criteria (FED0) and those with one ASD symptom criterion (FED1), a gradient of severity in AdAS Spectrum scores from CTL subjects to ASD patients, across FED0, ASD1, FED1 was shown. CONCLUSIONS: The AdAS Spectrum showed excellent internal consistency and test-retest reliability and strong convergent validity with alternative dimensional measures of ASD. The questionnaire performed differently among the three diagnostic groups and enlightened some significant effects of gender in the expression of autistic traits.

Glutamate receptor RNA editing in health and disease.

RNA editing is a post-transcriptional process with an important role in gene modification. This editing process involves site-selective deamination of adenosine into inosine in the pre-mRNA, leading to the alteration of translation codons and splicing sites in nuclear transcripts, thereby enabling functionally distinct proteins to arise from a single gene. One important instance is the neuron editing of the ionotropic glutamate receptors (iGluRs). GluRs play a key role in excitatory synaptic transmission and plasticity in the central nervous system (CNS); their channel properties are largely dictated by the subunit composition of the tetrameric receptors. AMPA/kainate channels are assembled from GluA1-4 AMPA or GluK1-5 kainate receptor subunits. In particular, three of the four AMPA and two of the five kainate receptor subunits are subject to RNA editing. The editing positions have been named on the basis of the amino acid substitutions, such as the Q/R site in AMPA GluA2; the Q/R site in GluK1 and GluK2; the R/G site in GluA2, GluA3, and GluA4; and the I/V and Y/C sites in GluK2. These amino acid changes lead to profound alterations of the channel properties. This paper reviews the most relevant data showing the importance of glutamate receptor RNA editing in finely tuning glutamatergic neurotransmission in the normal CNS and following alterations of the editing process in association with disease phenotypes. Overall, these data indicate that a highly regulated process of glutamate receptor editing is of key importance in the proper function of neuronal cells and in their ability to adapt and modulate synaptic function.

Psychopharmacological treatment in borderline personality disorder: A pilot observational study in a real-world setting.

Psychotherapy is the cornerstone of treatment for borderline personality disorder (BPD) while pharmacotherapy should be considered only as an adjunctive intervention. In clinical practice, however, most of BPD patients only receive medication. The aim of the study is to first describe pharmacological treatment in BPD patients in Italy and secondly to evaluate if comorbidity or illness severity are associated with the prescription of different class compounds. Data on pharmacological treatment and clinical evaluation of 75 BPD patients were collected in 5 clinical settings. The association between comorbidity and medication was assessed. Moreover, we evaluated the association between pharmacotherapy and severity, defined by a cluster analysis aimed at detecting different groups of patients. Most of the participants (82.7%) were characterized by polypharmacy, with a mean of 2.4 medications per person. Interestingly, the prescription didn't seem to depend on/be based on the severity of the disorder and was only partially determined by the presence of comorbidity. In conclusion, our findings are similar to what described in other clinical studies, supporting the idea that medication management for BPD is only partially coherent with international guidelines. This pilot study confirms the need for more rigorous studies to gain greater understanding of this topic and diminish the gap between guidelines and the real clinical world.

Association of partial AZFc region deletions with spermatogenic impairment and male infertility.

BACKGROUND: Complete deletions of the AZFc region in distal Yq are the most frequent molecular genetic cause of severe male infertility. They are caused by intrachromosomal homologous recombination between amplicons--large, nearly identical repeats--and are found in 5-10% of cases of azoospermia and severe oligozoospermia. Homologous recombination may also generate different partial deletions of AZFc, but their contribution to spermatogenic impairment has not been confirmed. METHODS: In this study we analysed the prevalence and characteristics of different partial AZFc deletions and their association with spermatogenic failure. We studied 337 infertile men with different spermatogenic impairment and 263 normozoospermic fertile men using AZFc specific sequence tagged site markers and DAZ specific single nucleotide variants. RESULTS: We identified 18 cases of partial AZFc deletions in the infertile group (5.3%) and one case in the control group (0.4%). Seventeen deletions had the "gr/gr" pattern, one the "b2/b3" pattern, and one represented a novel deletion with breakpoints in b3 and b4 amplicons. Partial AZFc deletions were associated with different spermatogenic phenotypes ranging from complete azoospermia to only moderate oligozoospermia. CONCLUSIONS: Together with published data, our analysis of DAZ gene copy suggested that the contribution of the different deletions to male infertility varies: only partial AZFc deletions removing DAZ1/DAZ2 seem to be associated with spermatogenic impairment, whereas those removing DAZ3/DAZ4 may have no or little effect on fertility. These data show that, beside complete AZFc deletions, specific partial deletions represent a risk factor for male infertility, even if with different effect on spermatogenesis.

Relationship between multiple forms of plasminogen activator in human breast tumors and plasma and the presence of metastases in lymph nodes.

Plasminogen activators (PAs), a family of proteases active in blood coagulation, may play an important role in cancer. Indeed, blood coagulation disorders, such as altered fibrinogen and fibrin metabolism and increased incidence of vascular thrombosis, are common in patients with advanced malignant disease. Different types of human tumors are known to contain high levels of PA. The isoelectric focusing patterns of the PAs present in tumors and plasma from patients with breast cancer were compared with those of purified human urokinase and melanoma tissue PA. The pattern of isoelectric molecular forms of PA active at pH 8 showed two groups of several bands: in plasma from tumor-bearing patients and controls, these groups were in the pl ranges of 6.6 to 6.8 and 8.0 to 8.5; in mammary adenocarcinoma tissue, the ranges were 6.8 to 7.9 and 9.0 to 9.4. These patterns were different from those obtained with purified markers; the latter were 5.8 to 9.4 and 5.9 to 7.6 for purified human urokinase and melanoma plasminogen tissue activator, respectively. PA activity in tumor-bearing patients was very high in malignant tissue and, on the contrary, very decreased in plasma; this latter decrease was correlated with the presence of metastases in the axillary lymph nodes. These results suggest that the high PA activity in the tumor tissue might participate in the destruction of the peritumoral tissue, thus allowing its invasion by tumor cells, whereas the low activity of PA in the plasma might increase plasma fibrin, reflecting thus an early disorder in blood coagulation which would enhance the formation of metastases.

Relationship between circulating plasminogen activators and tumor development in mice.

Plasminogen activators (PAs) present in the plasma of BALB/c mice and produced in vitro by murine tumor cell cultures (B77-3T3, SR-BALB, AA6) have been characterized using electrophoretic-zymographic techniques. BALB/c mouse plasma contains a main PA activity with an approximate molecular weight of 88,000 and pI 6.3, inhibited by anti-human tissue-type plasminogen activator (t-PA) serum, here defined as murine t-PA. On the contrary, all tumor cells tested release a PA activity with a molecular weight of 44,500 and pI 9.2 characteristic of urokinase-type activator (murine u-PA). The injection s.c. of the different tumorigenic cells into BALB/c mice leads to tumor development and to a rapid increase of t-PA from the first day following the injection. This early enhancement of t-PA activity is not detectable in mice given injections of lethally irradiated B77-3T3 cells. Moreover the development of the tumor in the animals is related to the appearance of increasing levels of u-PA in the blood. This activity is detectable in the plasma of treated mice almost 2 wk before detection of a tumor 1 mm in diameter. During tumor development, the molecular weight of the u-PA and t-PA forms present in the plasma does not change, while there is a decrease of the isoelectric point of the u-PA leading to the appearance of distinct PA activities with pI 7.6 and 8.9. Syngenic and allogenic lymphocytes, injected in BALB/c mice, do not induce any modification in the pattern of the plasma PA. The injection of highly metastatic cells, as opposed to nonmetastatic or low-metastatic cells, does not give rise to detectable levels of u-PA in the plasma of treated mice. These data suggest that the lack of plasma u-PA activity facilitates the formation of metastates, while the increase of this activity is important in tumor development and is independent of the metastatic potential of the injected cells.

Expression of urokinase-type plasminogen activator (u-PA), u-PA receptor, and tissue-type PA messenger RNAs in human hepatocellular carcinoma.

Expression of plasminogen activators (PAs) and urokinase-type PA receptor (u-PAR) is associated with tumor growth and invasion. For in vivo human tumor tissues, there is no information on gene expression of PAs in hepatocellular carcinoma (HCC) or other hepatic pathophysiological conditions. In this study we examined the relative levels of u-PA, tissue-type PA (t-PA), and u-PAR mRNA expression in human HCC by reverse transcription-PCR compared with those expressed in peritumoral hepatic tissues. Twenty-five of 25 HCCs expressed u-PA mRNA, as well as 16 of 25 hepatic peritumoral tissues. However, none of the 14 cases of nontumorous liver samples (i.e., normal parenchyma, steatosis, and nonspecific reactive and chronic hepatitis) showed detectable levels of u-PA mRNA. The same samples analyzed for uPAR and t-PA mRNAs exhibited higher levels of these mRNAs in the malignant tissues compared with nontumorous ones. A strong correlation was found between the relative levels of u-PA and t-PA mRNAs detected in the tumor and in the corresponding peritumoral tissues (P < 0.001 for u-PA; P < 0.02 for t-PA). However, there was no correlation between the expression of u-PA and t-PA in HCC (P = 0.565). Furthermore, a significant inverse correlation was found between survival months of male patients and the relative level of u-PA mRNA (P < 0.05) detected at the time of biopsy, whereas no correlation was found in the case of t-PA mRNA. These results are in line with the possible differential biological role of u-PA and t-PA in the tumor etiopathogenesis and suggest that the detection of relative levels of u-PA mRNA may be a useful prognostic factor for male HCC patients.

A family of fibronectin mRNAs in human normal and transformed cells.

Previously, two fibronectin mRNAs, generated by alternative splicing of the extra domain (ED) and type III connecting segments (IIICS) sequences, have been described in a human transformed cell line and in human liver, respectively. We now report on a family of fibronectin mRNAs identified by Northern blotting analysis in two normal human fibroblast strains (HEL 299 and Flow 7000) and five transformed cell lines (8387 and HT-1080, fibrosarcomas; G-361, melanoma; JEG-3, choriocarcinoma; and RD, rhabdomyosarcoma). Seven different fibronectin mRNA forms with electrophoretic mobilities ranging between 8.6 and 7.7 kb were identified. Each cell line contains three (HEL 299, Flow 7000 and 8387) or two (HT-1080, G-361, JEG-3 and RD) fibronectin mRNAs species with characteristic size. In all cell lines we detected one fibronectin mRNA form which lacks the ED sequence (ED- fibronectin mRNA) and one or two fibronectin mRNAs containing it (ED+ fibronectin mRNA). These data show that the presence of ED+ and ED- fibronectin mRNAs is a general feature of all cells tested. Moreover, the fibronectin mRNA pattern is characteristic of the cell type analyzed, suggesting the occurrence of specifically programmed splicing mechanisms in each cell line.

Cytogenetic analogy between myelodysplastic syndrome and acute myeloid leukemia of elderly patients.

The biological and clinical importance of cytogenetic analysis in myelodysplastic syndrome (MDS) and in acute myeloid leukemia (AML) is being increasingly recognized. Recently, cytogenetic similarities were noted between elderly de novo AML and secondary AML, suggesting common etiopathogenetic mechanisms. In the present study we analyzed the cytogenetic similarities between patients with AML of different age and patients with MDS consecutively diagnosed during a 5-year period at a single, primary referral, hematologic center. Of 246 patients aged <86 years, 195 (80%) had a cytogenetic study at diagnosis. Informative metaphases were obtained in 182 cases (93%), including 17 (9.3%) with secondary MDS/AML. Patients were classified according to FAB criteria and were subdivided into four groups: (1) 'early MDS': 42 patients with MDS of FAB subtypes other than refractory anemia with excess of blasts (RAEB) or RAEB in transformation (RAEB-T); (2) 'late MDS': 35 patients with RAEB and RAEB-T; (3) 'old AML': 48 patients with AML aged 65 to 85 years; (4) 'young AML': 57 patients with AML aged <65 years. Results showed that 'late MDS' and 'old AML' had striking cytogenetic similarities both in the frequency of normal karyotypes (31% and 27%), single abnormalities (14% and 13%), double abnormalities (17% and 14%), complex karyotypes (37% and 46%), and numerical abnormalities (89% and 93%), as well as in the frequency of rearrangements involving chromosome 5 (20% and 31%) and 7 (27% and 27%). The only difference between the two groups was found in the median number of chromosomes involved in complex karyotypes (5 vs 8; P=0.03). 'Early MDS' had significantly less complex karyotypes (21%; P<0.05), but its cytogenetic features resembled otherwise those of 'late MDS' and 'old AML', and any significant difference disappeared when patients with chronic myelomonocytic leukemia (CMML) were excluded. CMML markedly differed from other MDS subtypes in the frequency of normal (57%) and of complex karyotypes (6%). Secondary MDS/AML and AML with trilineage dysplasia shared the same cytogenetic features of 'late MDS' and 'old AML'. 'Young AML' strikingly differed from all other groups, particularly in the higher frequency of balanced translocations (29%; P<0.001) and single karyotype abnormalities (32%; P<0.02), and in the lower frequency of complex karyotypes (19%; P<0.01) and of chromosome 5 (2%; P<0.001) and 7 (9%; P<0.01) involvement. We conclude that in a population-based series of patients, the cytogenetic profile of MDS, particularly of RAEB/RAEB-T, was nearly identical to that of elderly patients with AML both in the frequency and in the type of chromosomal abnormalities. These results support the possibility that MDS and AML of elderly patients may represent the same disease seen at different stages of evolution.

Homozygosity mapping of a gene for arterial tortuosity syndrome to chromosome 20q13.

BACKGROUND: Arterial tortuosity syndrome (ATS) is an uncommon connective tissue disorder of unknown aetiology. The most prominent feature is tortuosity of the large arteries, but lengthening, stenosis, and aneurysm formation are also frequent. METHODS: We performed a genomewide screen by homozygosity mapping of three consanguineous multiplex families, two from Morocco, and one from Italy, which included 11 ATS patients. The two families from Morocco may possibly have a common ancestor. RESULTS: We mapped the ATS gene to chromosome 20q13. Recombinations within an extended haplotype of 11 microsatellite markers localised the ATS gene between markers D20S836 and D20S109, an interval of 9.5 cM. CONCLUSIONS: Cloning and completing functional and structural analysis of the ATS gene may provide new insights into the molecular mechanisms of elastogenesis.

RNA editing: a molecular mechanism for the fine modulation of neuronal transmission.

The term "RNA editing" is used to identify any mechanism responsible for producing mRNA molecules with sequence information not specifically encoded in the DNA. RNA editing is therefore an important event in gene modification, which takes place at a post-transcriptional level. The molecular mechanism of RNA editing involves site-selective deamination of adenosine to inosine in pre-mRNA, which leads to altering translation codons and splicing in nuclear transcripts, whereby functionally distinct proteins can be produced from a single gene. The mammalian editing enzymes ADARs (adenosine deaminases acting on RNA) are widely expressed in brain and other tissues: however, up until now their substrates have mainly been found in the Central Nervous System (CNS). Of particular relevance in the CNS is the editing occurring at the ionotropic glutamate receptors (GluRs) level. Three AMPA and two Kainate receptors are subject to RNA editing. The consequence of this process is the substitution of specific amino acids in functionally critical positions of the receptors. Depending on the GluR involved, the consequences of editing will involve: activation and/or inhibition of splicing sites; modulation of the trafficking of the receptor to the plasma membrane; the process of tetramerization of the receptor subunits; modification of the ions passage through the receptor channel; modulation of the desensitization and action potential recovery times. All these events are specific to the different GluRs and are genetically and developmentally controlled. RNA editing is therefore a crucial event involved in controlling transmission of the action potential at the postsynaptic level. This modulation involves the transmission of all sensory stimuli to the CNS and gives rise to the "Sensotype". The Sensotype therefore defines the "way" in which the information acquired from the environment by the sensory systems is transmitted to the brain. The signals and inputs deriving from the Sensotype are transmitted to the brain, which processes and stores these signals thus generating the "Brainotype". Brainotype and Sensotype are genetically and environmentally determined; they are individually unique and specific to every living organism with a nervous system. Their characteristics are, at least in part, dependent on the modulation of the "RNA editing" process since glutamate receptors represent the main neurotransmitter system in the CNS.

A -96C-->T mutation in the promoter of the collagen type VII gene (COL7A1) abolishing transcription in a patient affected by recessive dystrophic epidermolysis bullosa.

Hereditary dystrophic epidermolysis bullosa (DEB) refers to a group of clinically heterogeneous skin blistering diseases due to mutations in the collagen type VII gene (COL7A1). We report two novel mutations found in a patient affected by the most severe form of DEB, the recessive Hallopeau-Siemens variant (HS-RDEB): the R1978X nonsense mutation, in exon 72, and the -96C-->T transition, in the promoter region. The allele specific analysis of the transcripts from skin fibroblasts of the patient showed that the -96C-->T mutation is associated to the absence of collagen type VII (COLVII) mRNA. This mutation, the first one ever identified in the promoter of COL7A1, falls in an Sp1 motif, localized between nucleotides -96 and -91. Gel shift analysis indicated that the -96/-91 sequence is a functional Sp1 site and that the -96C-->T transition inhibits the binding of the trascription factor. These data indicate that the -96/-91 sequence is a crucial Sp1 site whose integrity is necessary for COL7A1 expression. The compound heterozygosity for the -96C-->T null mutation and for the R1978X mutation leads to the absence of COLVII at skin level and to the severe phenotype of the HS-RDEB patient. Hum Mutat 16:275, 2000.

Degradation of human plasma and extracellular matrix fibronectin by tissue type plasminogen activator and urokinase.

Fibronectins and plasminogen activators, both tissue and urokinase types, are involved in the physiopathological degradation of the extracellular matrix. Previous reports indicate that fibronectin can be degraded by urokinase without plasminogen. Also, we have shown that tissue-type plasminogen activator can cleave fibronectin, without plasminogen, generating fragments of 30 and 220-250 kDa detectable by immunoblotting analysis. A comparison with urokinase-induced degradation indicates that the cleavage sites are the same for both plasminogen activators. One is close to the carboxyl-terminal, disrupting the fibronectin dimeric structure, and one is near the amino-terminal, generating a 30 kDa fragment. In solution, the activity of tissue-type plasminogen activator was prevalent on the amino-terminal site, while urokinase activity was prevalent on the carboxyl-terminal site. On fibronectin immobilized onto a gelatin coated surface, only the 30 kDa fragment was released when treated with both plasminogen activators. Plasminogen activators also were active on fibronectin assembled into the extracellular matrix of cultured fibroblasts. Urokinase caused the complete disappearance of extracellular matrix fibronectin, together with the release of the 30 and the 220-250 kDa fibronectin fragments, but left a flat morphology, while tissue-type plasminogen activator induced the release of the 30 kDa fragment associated with changes in cellular morphology. The plasminogen-independent fibronectin degradation exerted by tissue-type plasminogen activator and urokinase is 100 times lower than that exerted by plasmin. This may provide a mechanism for localized and limited degradation of fibronectin preventing the generalized proteolysis associated with plasminogen activation.

Genomic organization of the human GRIK2 gene and evidence for multiple splicing variants.

Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by L-glutamate. Here we present the genomic organization of the human GRIK2 gene, which codes for the kainate GluR6 receptor subunit, deduced from sequence data present in the public databases and analyzed by bioinformatic tools. By similarity search using the human GluR6 cDNA sequence against non-redundant databases, we found three positive entries (AP002528, AP002529, and AP002530 deposited by Hirakawa et al., 2000) which are part of a BAC contig of about 1 Mb spanning region 6q21. The GRIK2 gene was found to be split into 17 exons, covering about 670 kb of the region. The availability of the data on the genomic organization allowed the study of GRIK2 gene expression by RT-PCR analysis which was performed on human teratocarcinoma cell cultures (NT2) and on mRNA obtained from human hippocampus (Clontech). The study gives evidence for several different splicing variants in addition to the previously cloned human GluR6 cDNA (ID: U16126). The splicing mechanism leading to the different isoforms involves exons 11, 12 and 16. The mRNA containing exon 16 at the 3' end is the homolog to the mouse GluR6-2. The translation of this mRNA would code for a different intracellular C-terminus, as compared to that coded by the known human isoform. The newly identify isoform is the predominant form expressed in human teratocarcinoma NT2 cells. All the newly identified mRNAs isoforms are expressed in NT2 cells and in human hippocampus mRNA at variable levels and would be responsible for the production of five different putative GluR6 receptor subunits, some differing in the C-terminal domains (mouse homolog) and some lacking specific transmembrane domains.

Identification of novel alternatively-spliced mRNA isoforms of metabotropic glutamate receptor 6 gene in rat and human retina.

Novel splice variants of metabotropic glutamate receptor type 6 (mGlu6 receptor) were identified by reverse transcription-polymerase chain reaction (RT-PCR) amplification and sequence analysis of rat and human retina cDNAs. The new rat mGlu6 receptor mRNA isoform is characterized by an additional exon of 88 nucleotides containing an in frame stop codon, thus predicting the expression of a truncated protein of 508 amino acids. The human retina was found to express two different mGlu6 receptor mRNA variants: one lacking 97 nucleotides from exon 6, the other including five nucleotides of intron 5. These mRNAs would encode truncated receptors of 425 and 405 amino acids, respectively. Both in rats and in humans, the truncated mGlu6 receptor proteins would comprise the extracellular domain but lack the transmembrane and intracellular portion of the receptor, thus possibly acting as retinal soluble receptors for glutamate. Though generated by different patterns of alternative splicing, the inter-species conservation of truncated mGlu receptor molecules strongly suggest their relevance in the regulatory network of glutamatergic neurotransmission.

Genomic organisation and chromosomal localisation of the gene encoding human beta adducin.

Adducin (ADD) is a heterodimeric protein of the membrane skeleton with subunits of 103 (alpha) and 97 kDa (beta). It promotes the assembly of the spectrin-actin network. We have previously shown that one point mutation in each of the alpha and beta rat ADD-encoding genes is associated with blood pressure variation in an animal model for hypertension, the Milan hypertensive strain of rats, probably due to a change in the phosphorylation pattern. In fact, the rat mutations, Y to F for alpha and R to Q for beta, are located, respectively, in a tyrosine kinase and a protein kinase A phosphorylation site. We have now determined, for the human beta-ADD-encoding gene, its chromosomal localisation, exon-intron organisation and alternative splicing patterns. We report here that human beta-ADD is localised on chromosome 2 and we also show a characteristic 3' end alternative splicing of the beta-ADD RNA that generates two distinct beta-ADD families, namely ADD 63 and 97; both of them in turn present a very complex differential splicing pattern in the internal exons.

Phosphorylation of human plasminogen activators and plasminogen.

Plasminogen (PG), urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) are the main molecules involved in fibrinolysis and in many other physiological and pathological processes. In the present study we report that human t-PA, purified from human melanoma cells, and PG, purified from human plasma, both contain P-Tyr residues, as revealed by immunoblotting analyses with monoclonal anti-P-Tyr antibodies. In addition HPLC amino acid analysis of acid-hydrolyzed t-PA, PG and u-PA, shows that: (i) P-Ser and P-Tyr residues are present in t-PA; (ii) P-Thr and P-Tyr are present in PG; (iii) P-Ser, P-Thr and P-Tyr are present in u-PA. The utilization of monoclonal anti-P-Ser and anti-P-Thr antibodies in immunoblotting experiments has confirmed these data which indicate that phosphorylation is a common feature of PAs and of PG.

Tyrosine phosphorylation of human urokinase-type plasminogen activator.

Immunoblotting analysis of purified human urokinase plasminogen activator (u-PA), gives a positive signal when reacted with anti-phosphotyrosine monoclonal antibodies (MoAb anti-P-Tyr); competition with o-phospho-DL-tyrosine (P-Tyr) but not o-phospho-DL-threonine or serine (P-Treo, P-Ser) completely suppresses this signal. Either the 55 kDa u-PA form and the lower Mw form (33 kDa) derived from the 55 kDa u-PA are Tyr-phosphorylated also the u-PA secreted in the culture media of human fibrosarcoma cells (HT-1080) is phosphorylated in tyrosine as well as u-PA present in tissue extracts of tumors induced in nude mice by HT-1080 cells. These data show that urine purified human u-PA and u-PA produced by human fibrosarcoma cells, in vitro and in vivo, are phosphorylated in tyrosine; furthermore our data show that u-PA is the major Tyr-phosphorylated protein present in these human tumor cells.

Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization.

Mucolipin-1 is a 65-kDa membrane protein encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We studied the subcellular localization of wild-type and three different mutant forms (T232P, F408del and F465L) of mucolipin by expressing Myc-tagged proteins in HeLa cells. The overexpressed wild-type mucolipin colocalizes to late endocytic structures and induces an aberrant distribution of these compartments. F408del and F465L MLIV mutant proteins show a distribution similar to the wild-type protein, whereas T232P is retained in the endoplasmic reticulum. Among the mutants, only F408del induces a redistribution of the late endocytic compartment. These findings suggest that the overexpression of the mucolipin cation channel influences the dynamic equilibrium of late endocytic compartments.