Signaling by phosphoinositide-3,4,5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains.

Signal transmission by many cell surface receptors results in the activation of phosphoinositide (PI) 3-kinases that phosphorylate the 3' position of polyphosphoinositides. From a screen for mouse proteins that bind phosphoinositides, the protein GRP1was identified. GRP1 binds phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4, 5)P3] through a pleckstrin homology (PH) domain and displays a region of high sequence similarity to the yeast Sec7 protein. The PH domain of the closely related protein cytohesin-1, which, through its Sec7 homology domain, regulates integrin beta2 and catalyzes guanine nucleotide exchange of the small guanine nucleotide-binding protein ARF1, was also found to specifically bind PtdIns(3,4,5)P3. GRP1 and cytohesin-1 appear to connect receptor-activated PI 3-kinase signaling pathways with proteins that mediate biological responses such as cell adhesion and membrane trafficking.

Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata.

A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of (3)H2 to double bond of 3,4-dehydroproline residue. (3)H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl beta-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [(3)H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[(3)H] Pro-Ala-OH, [(3)H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[(3)H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[(3)H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro(5)-[(3)H] Pro-OH and H-Asp-[(3)H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.

[Drug consumption and risks of polypharmacotherapy in elderly population]. / Spotreba léciv v seniorské populaci a rizika polyfarmakoterapie ve stárí.

BACKGROUND: One of our previous studies was aimed at the consumption of prescribed drugs by the elderly population. The average per day number of drugs was 4.6 (maximum 13). Existence of freely obtainable drugs with massive advertisements brings a question, how many of those drugs it is necessary to add in order to estimate probability of interaction and undesirable drug effects. In order to achieve valid information, students of the sixth year of General medicine program during their practical course at general practitioners were asked to interview randomly selected senior patients. They asked on the number, type, and price of freely obtainable drugs which they use. Data were evaluated from interviews accomplished during academic years 2001/2002 and 2004/2005. METHODS AND RESULTS: Our cohort included 252 men and 148 women with average age of 78.7 years. Average number of freely obtainable drugs was 2.26 at the beginning and 2.32 at the end of study. Only 34% of questioned did not buy any of those drugs at all or only exceptionally, 66% reported buying once a month or weekly. 44% of seniors buy analgetics, 58% buy vitamins, 37% food supplements, 36% non steroid antirheumatics, 46% cold prevention drugs, 30% anti-constipation drugs. Contrary to our expectation, positive correlation between the sums given for the personal participation on the drug costs and that given for freely obtainable drugs was found. It is not possible to expect, that polymorbidic patient with several prescribed drugs would buy less of freely obtainable drugs even due to the financial requirements. CONCLUSIONS: Freely obtainable drugs, many of them composites, can represent significant source of interactions and undesirable drug effects. They can also significantly modulate compliance of the senior. The high percentage of seniors buying freely obtainable drugs requires aimed questions on the pharmacological history.

Genetic mapping of adrenergic receptor genes in humans.

We have genetically mapped the genes encoding four human adrenergic receptors (ARs) of subtypes alpha 1C, alpha 2A, alpha 2B, and beta 1, which are prototypic G protein coupled receptors that mediate the physiological effects of neurotransmitters, hormones, and drugs. We placed these genes onto the Cooperative Human Linkage Center (CHLC) and Genethon framework maps, within confidence intervals with greater than 1000:1 odds. With multipoint analysis the alpha 1C gene (locus ADRA1C) mapped to the interval between NEFL and D8S283; alpha 2-C4, the gene encoding the alpha 2C AR (locus ADRA2C), mapped to the interval between D4S126 and D4S62; and the alpha 2-C10 (alpha 2A AR)/beta 1 haplotype (loci ADRA2A/ADRB1) mapped to the interval between D10S259 and D10S187. A fifth AR gene, beta 2, yielded significant LOD scores with markers on the long arm of chromosome 5; however, this locus (ADRB2) could not be mapped to any specific interval with odds of greater than 1000:1. The two AR genes that are completely linked, alpha 2-C10 and beta 1, were oriented on their shared 225-kb genomic fragment relative to the direction of transcription, with beta 1 being 5' to alpha 2-C10. The positioning of these genes on high-density framework maps allows them to be tested as candidates in a spectrum of diseases that might involve AR dysfunction.

Analysis of chromosome 18 DNA markers in multiplex pedigrees with manic depression.

Six pedigrees segregating manic-depressive illness (MDI) were analyzed for linkage to 21 highly polymorphic microsatellite DNA markers on chromosome 18. These markers span almost the entire length of the chromosome, and gaps between markers are less than 20 cM. In particular, we analyzed several markers localizing to the pericentromeric region of chromosome 18 which generated lod scores suggestive of linkage in an independent study. Lod score analysis was performed and results were examined by family. One region produced positive lod scores, though at 18q23 and not in the pericentromeric region. We additionally used two nonparametric methods because the true mode of transmission of MDI is unknown; results were again somewhat suggestive for markers in the region of 18q23 but not in the pericentromeric region.

Use of a neurophysiological trait in linkage analysis of schizophrenia.

Traditional diagnostic techniques may not provide all the information necessary to reveal the genetic causes of schizophrenia through linkage analysis. Use of neurophysiological indicator variables that are associated with the disease may increase the probability of detecting linkage. Such variables not only produce simpler phenotypes for analysis, but they also may be more proximal to the gene products involved in neurological dysfunctions underlying schizophrenia. We have used a previously characterized neurophysiological variable, the P50 evoked-auditory response, to search for chromosomal regions that may be of interest in the study of schizophrenia. Although our scan of over 300 markers did not show strong evidence for linkage to P50 in nine families, this exploratory analysis has revealed several chromosomal regions that may deserve further study.

Search for a gene predisposing to manic-depression on chromosome 21.

Six kindreds containing multiple cases of Manic-depressive illness (MDI) were genotyped with seven highly polymorphic microsatellite loci used in the construction of an index map for chromosome 21. The kindreds were also genotyped with a microsatellite polymorphism for PFKL, a chromosome 21 locus that has shown suggestive linkage to MDI in one pedigree [Straub et al., 1993: The American Society of Human Genetics]. Evidence of linkage was not found assuming either autosomal dominant or recessive inheritance. The nonparametric affected sib pair test did not yield significant evidence of linkage.

Mutation scan of the D1 dopamine receptor gene in 22 cases of bipolar I disorder.

In a previous study [Coon et al., Am J Hum Genet 52:1234-1249, 1993], we found suggestive evidence of linkage between manic-depressive illness (MDI) in eight multiplex pedigrees and D5S62, a DNA marker mapping to the telomeric region of 5q. As the D1 dopamine receptor gene (DRD1) maps to this region and as alterations in dopaminergic neurotransmission have been indirectly implicated in the pathogenesis of MDI, we directly searched for mutations in the coding region of the DRD1 gene in 22 unrelated cases of bipolar I (BPI) disorder derived from multiplex families. Using single strand conformation polymorphism (SSCP) analysis, we did not observe any abnormal SSCP variants in the BPI cases that differed from controls.

Search for a schizophrenia susceptibility locus on human chromosome 22.

We used 10 highly informative DNA polymorphic markers and genetic linkage analysis to examine whether a gene locus predisposing to schizophrenia is located on chromosome 22, in 105 families with schizophrenia and schizoaffective disorder. The LOD score method, including analysis for heterogeneity, provided no conclusive evidence of linkage under a dominant, recessive, or penetrance free model of inheritance. Affected sib-pair analysis was inconclusive. Affected pedigree member analysis gave only suggestive evidence for linkage. Multipoint APM analysis, using 4 adjacent loci including D22S281 and IL2RB, a region of interest from the APM analysis, gave non-significant results for the three different weighting functions.

C to T nucleotide substitution in codon 713 of amyloid precursor protein gene not found in 86 unrelated schizophrenics from multiplex families.

Jones et al. Nature Genet 1:306-309, [1992] recently detected a C to T nucleotide transition (codon 713) in a highly conserved region of the beta-amyloid precursor gene in a single case of schizophrenia. Although the sequence variant may be a natural polymorphism, it is crucial to determine whether the mutation might be present in a small subset of schizophrenics. We isolated DNA from 86 unrelated chronic schizophrenics who had a first degree relative with chronic schizophrenia or chronic schizoaffective disorder. After PCR amplification of exon 17, we were unable to detect the presence of the codon 713 variant in these schizophrenic cases, as well as in 156 controls. Unless additional cases are found with the codon 713 mutation, it is unlikely that the sequence variant is pathogenic for schizophrenia.

Genomic scan for genes predisposing to schizophrenia.

We initiated a genome-wide search for genes predisposing to schizophrenia by ascertaining 9 families, each containing three to five cases of schizophrenia. The 9 pedigrees were initially genotyped with 329 polymorphic DNA loci distributed throughout the genome. Assuming either autosomal dominant or recessive inheritance, 254 DNA loci yielded lod scores less than -2.0 at theta = 0.0, 101 DNA markers gave lod scores less than -2.0 at theta = 0.05, while 5 DNA loci produced maximum lod scores greater than 1: D4S35, D14S17, D15S1, D22S84, and D22S55. Of the DNA markers yielding lod scores greater than 1, D4S35 and D22S55 also were suggestive of linkage when the Affected-Pedigree-Member method was used. The families were then genotyped with four highly polymorphic simple sequence repeat markers; possible linkage diminished with DNA markers mapping nearby D4S35, while suggestive evidence of linkage remained with loci in the region of D22S55. Although follow-up investigation of these chromosomal regions may be warranted, our linkage results should be viewed as preliminary observations, as 35 unaffected persons are not past the age of risk.

Analysis of chromosome 22 markers in nine schizophrenia pedigrees.

Previous results of a genome-wide survey for schizophrenia susceptibility genes in nine multiplex families indicated a possible region of linkage on chromosome 22. We therefore tested for linkage using ten highly polymorphic chromosome 22 DNA markers. Lod score analyses were suggestive of linkage for several markers on the distal end of the chromosome; however, no lod score exceeded 3 assuming either autosomal dominant or autosomal recessive transmission. The highest lod score was 2.09 (theta = 0.10) for marker D22S276 under autosomal recessive inheritance. Based on simulation analyses, this result is unlikely to represent a false positive. Analyses using information from affected individuals only resulted in reduced lod scores, with a maximum of 1.40 (theta = 0.05) for D22S276 assuming autosomal recessive inheritance. Two nonparametric methods, sib pair analysis and the Affected-Pedigree-Member method, also yielded suggestive but inconclusive findings; results were positive, but strict thresholds of significance were not met. Additionally, we tested one candidate gene, the Arylsulfatase A gene, located in the region of 22q13.31-qter. Results were again inconclusive, though the DNA marker available for this gene was a 2-allele RFLP with heterozygosity of 0.5, and therefore not maximally informative. Further investigation of this chromosomal region and this and other candidate genes may be warranted.

Search for mutations in the beta 1 GABAA receptor subunit gene in patients with schizophrenia.

As alterations in GABAergic neurotransmission have been indirectly implicated in the pathogenetics of schizophrenia, GABAA receptor subunit genes are plausible candidate genes for the illness. We undertook a search for sequence variations in the coding region of beta 1 subunit gene by designing intron-based primers to amplify its 9 exons. Using single strand conformation polymorphism (SSCP) analysis, we found an exon 9 variant present in 3 of 86 unrelated schizophrenic cases derived from families having at least 2 first-degree relatives with schizophrenia. Direct sequencing of the SSCP variant revealed a C-->G nucleotide transversion at codon 396 predicting a histidine to glutamine substitution in the beta 1 peptide. The predicted amino acid substitution occurs at a highly conserved site, 9 residues from a cAMP-dependent serine phosphorylation consensus sequence. All known GABAA beta 1 subunit genes including human, bovine, and rat, code for histidine at position 396. Although the variant cosegregated with disease in a family with 2 affected sibs, it was only transmitted to 2 of 3 affected sibs in a multiplex family. The variant was not found in an additional sample comprising 155 unrelated schizophrenics and the sequence variant was present at a low frequency (approximately 1.1%) in control groups. Although these results indicate that the sequence variant is likely to be a natural polymorphism, it is possible that the variant may be a predisposing allele in rare instances. It is also possible that the variant may change the function or regulation of the GABAA receptor complex and it may be of pharmacogenetic relevance.

A linkage study with D5 dopamine and alpha 2C-adrenergic receptor genes in six multiplex bipolar pedigrees.

Six kindreds containing multiple cases of manic-depressive illness were genotyped with highly polymorphic microsatellite polymorphisms for the D5 dopamine and alpha 2C-adrenergic receptor genes. Evidence of linkage was not found assuming either autosomal dominant or recessive transmission. The non-parametric sib pair test did not yield evidence of linkage.

Anticipation in multiplex schizophrenia pedigrees.

Anticipation, a phenomenon in which the symptoms of an inherited disease become more severe and age of onset occurs earlier across generations, has become an issue of importance in schizophrenia. If anticipation is found in families manifesting the illness, a possible type of genetic mutation would be implicated for predisposition to schizophrenia, as anticipation is now known to result from expansion of unstable trinucleotide repeat sequences. Two recent studies have tested for evidence of this phenomenon in schizophrenia families and came to differing conclusions; it is possible that anticipation occurs only in a subset of families. Our sample shows significant decreases in age of onset and increases in severity across generations. To investigate possible ascertainment bias, we looked at early onset parents and found no consistent anticipation effects in this group. We did find some increase in anticipation for mother-child vs father-child pairs; this possible imprinting effect may indicate true anticipation, though the evidence is not strong in our small sample, and the most conservative interpretation of our results is that the differences are due to ascertainment bias.

Schizophrenia and glutamate receptor genes.

Nine multiplex schizophrenia families were genotyped with polymorphisms for the GLUR5 and NMDAR1 glutamate receptor subunit genes. Using the lod score technique, evidence of linkage was not found assuming either dominant or recessive transmission. Similarly, the non-parametric sib pair test did not yield significant evidence of linkage.

Analysis of GABAA receptor subunit genes in multiplex pedigrees with manic depression.

The gamma-aminobutyric acid (GABA) neurotransmitter system has been implicated in the pathogenesis of manic depression. Tests of this hypothesis can now be carried out due to the recent characterization of simple sequence repeat polymorphisms for the GABAA receptor alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, beta 1, beta 3 and gamma 2 subunit genes. Using both parametric and non-parametric methods, we tested for linkage between manic depression and these polymorphisms in six multi-generational pedigrees. No evidence of linkage was found.

Linkage analysis between schizophrenia and a microsatellite polymorphism for the D5 dopamine receptor gene.

Using 23 multiplex pedigrees we tested for linkage between schizophrenia and a microsatellite polymorphism for the D5 dopamine receptor gene (DRD5). Assuming autosomal dominant inheritance and a maximum penetrance of 0.6, an overall lod score of -4.54 was derived at 0% recombination. For recessive transmission the summary lod score was -8.37 at 0% recombination. These data suggest that mutations of the D5 dopamine receptor gene are unlikely to be of major etiological importance in the pathogeneses of schizophrenia in the families studied. However, our study does not exclude the D5 dopamine receptor gene as a candidate gene for schizophrenia because some of our families were not informative for linkage and because of the likelihood of genetic heterogeneity.

Schizophrenia and GABAA receptor subunit genes.

Alterations in gamma-aminobutyric acid (GABA) neurotransmission have been indirectly implicated in the pathogenesis of schizophrenia. Using nine multiplex pedigrees, we tested for linkage between schizophrenia and simple sequence repeat polymorphisms for the GABAA receptor alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, beta 1 and beta 3 subunit genes. Evidence of linkage was not found when assuming either autosomal dominant or autosomal recessive inheritance. The non-parametric sib pair test also did not reveal significant evidence of deviation from expected segregation ratios.

Search for a schizophrenia susceptibility gene on chromosome 18.

Nine multiplex schizophrenia families were genotyped with 15 microsatellite markers mapping to the short and long arm of chromosome 18. Assuming either autosomal dominant or recessive inheritance evidence of linkage was not found. In addition, the non-parametric sib pair test did not reveal significant evidence of linkage.