Gregorio Seidita

Angiotensin-Converting Enzyme Gene Deletion Polymorphism Determines an Increase in Frequency of Migraine Attacks in Patients Suffering from Migraine without Aura

Many authors have reported an association between the angiotensin-converting enzyme (ACE)-D allele and coronary heart disease and other cardiovascular diseases. The mechanism underlying the positive associations between the ACE-D alleles and diseases are not yet clear. Previous reports showed an association between migraine without aura and ACE-D allele polymorphism. The study is aimed to evaluate if the DD genotype could also be associated with the frequency and duration of migraine without aura. In 302 patients suffering from migraine without aura (at least for 1 year), with no history of cardiovascular diseases and major risk factors for ischemic events, the genotypes of the ACE gene, plasma ACE activity, and the frequency (weekly) and duration of migraine attacks were evaluated. No drugs were given before (4 weeks) and during the study. The same evaluations were performed in 201 subjects without migraine. The molecular biologist and the physician evaluating the patient data were blinded to the clinical history and ACE-DD gene determination. Genotypes were determined by polymerase chain reaction amplification. Plasma ACE activity was performed by the HPLC method. The groups were similar for sex, age and smoking habit (migraines: 302 patients (200 F/102 M), mean age 37.8 ± 8.2 years; control: 201 subjects (127 F/74 M), mean age 37.5 ± 9.3 years). Patients with migraine without aura showed higher incidence of the ACE-DD gene (48.34%) than control subjects (37.32%), p < 0.05. The frequency of migraine (average attacks per week) was higher in patients with DD (2.11 ± 1.9) than in patients with ID (1.54 ± 1.44), p < 0.05. No difference in duration of migraine attacks (hours per week) was observed. Plasma ACE activity was increased in patients with the ACE-DD gene. Our data suggest that ACE-DD gene polymorphism could have an important role in determining migraine attacks and the frequency of these attacks. Further data are needed through further studies, especially on the biomolecular level.

RAS residues that are distant from the GDP binding site play a critical role in dissociation factor-stimulated release of GDP.

We have previously shown that a conserved glycine at position 82 of the yeast RAS2 protein is involved in the conversion of RAS proteins from the GDP‐ to the GTP‐bound form. We have now investigated the role of glycine 82 and neighbouring amino acids of the distal switch II region in the physiological mechanism of activation of RAS. We have introduced single and double amino acid substitutions at positions 80–83 of the RAS2 gene, and we have investigated the interaction of the corresponding proteins with a yeast GDP dissociation stimulator (SDC25 C‐domain). Using purified RAS proteins, we have found that the SDC25‐stimulated conversion of RAS from the GDP‐bound inactive state to the GTP‐bound active state was severely impaired by amino acid substitutions at positions 80–81. However, the rate and the extent of conversion from the GDP‐ to the GTP‐bound form in the absence of dissociation factor was unaffected. The insensitivity of the mutated proteins to the dissociation factor in vitro was paralleled by an inhibitory effect on growth in vivo. The mutations did not significantly affect the interaction of RAS with adenylyl cyclase. These findings point to residues 80–82 as important determinants of the response of RAS to GDP dissociation factors. This suggests a molecular model for the enhancement of nucleotide release from RAS by such factors.

Migraine Without Aura and ACE-Gene Deletion Polymorphism: Is There a Correlation? Preliminary Findings

Dear Sir, There has been a surge of research on the angiotensinconverting enzyme (ACE) gene deletion polymorphism, and there is continuing interest in the link between a variant deletion of the gene for ACE and an increased incidence of myocardial infarction [1]. Although some studies have failed to confirm this finding [2], many have reported an association between the ACE D allele and coronary heart disease (CHD) [3–5] and other cardiovascular diseases [6,7]. The mechanisms underlying the positive associations between the ACE-D alleles and diseases are not yet clear. There is increasing evidence linking the reninangiotensin-aldosterone system with the regulation of cardiac and vascular growth. ACE circulates in the plasma and is present on the surface of endothelial cells, where it stimulates the conversion of the inactive angiotensin I to the highly active angiotensin II. Angiotensin II is a potent vasoconstrictor. An attractive hypothesis regarding this is that the effects of the ACE-D allele polymorphism are mediated by altered expression of tissue and/or circulating ACE. To date there have not been any reports on the association between migraine without aura (IHS 1988) and ACED allele polymorphism. We evaluated the genotypes of the ACE gene in 191 subjects (130 females and 61 males), with a mean age 37.8 6 8.4 (range 25–46) years, suffering from migraine without aura for at least for 1 year and 201 subjects who did not have a history of headache (123 females and 74 males), with a mean age 37.5 6 9.3 (range 26–46) years. DNA was prepared from a small aliquot of whole blood collected in ethylenediaminetetraacetic acid (EDTA) by using a DNA extraction matrix. Genotypes were determined by polymerase chain reaction amplification of the I/D region of the ACE gene using oligonucleotide primers and resolving the amplified products on 1.5% agarose gels containing ethidium bromide, with modifications to prevent mistyping of I/D individuals as DD homozygotes (as previously reported) [2]. Blank controls were routinely included with each set of amplifications to exclude contamination. Genotypes were determined without knowledge of the case control status and, as a further check, 30% of subjects were chosen to undergo repeat genotyping using freshly prepared DNA, and all cases were confirmed. Only patients with no family history of cardiovascular diseases and major risk factors for ischemic events were included in the study. DD of the ACE gene was shown to be more common in subjects with a history of migraine without aura (DD 5 49.7%, ID 43.5%, II 6.8%) than in those with it (DD 37.5%, ID 50.5%, II 12%); (p , 0.05). Our data suggest that the DD genotype could be a strong independent discriminator of migraine without aura. Moreover, these data agree with our previous double-blind randomized study, which showed that captopril was effective in reducing the frequency, duration, and severity of migraine without aura paroxysms [8]. Our findings are clinically significant. The large incidence of ACE-DD in patients with migraine without aura suggests the possibility of treating patients with migraine without aura with ACE inhibitors. In fact, captopril has been shown to restore the availability of endogenous opioids, to improve cerebral flow by metabolism of both the cerebral and systemic reninangiotensin system or by catecholamine release, and to restore the nociceptive–antinociceptive balance through an increase in bradykinin levels [9]. In addition, bradykinin is a vasodilator that inhibits vascular smooth muscle cell proliferation and can stimulate the release of endothelial vasodilators, including nitric ox-

Lack of association of HOXA1 and HOXB1 mutations and autism in Sicilian (Italian) patients

Lack of association of HOXA1 and HOXB1 mutations and autism in Sicilian (Italian) patients

Cloning and sequencing of the dnaK region of Streptomyces coelicolor A3(2)

The dnaK homologue of Streptomyces coelicolor A3(2) strain M145 has been cloned and sequenced. Nucleotide sequence analysis of a 2.5-kb region revealed an open reading frame (ORF) encoding a predicted DnaK protein of 618 amino acids (M(r) = 66,274). The dnaK coding sequence displays extreme codon bias and shows a strong preference for CGY and GGY, for Arg and Gly codons, respectively. The predicted DnaK sequence has a high Lys:Arg ratio which is not typical of streptomycete proteins. The region immediately downstream from dnaK contains an ORF for a GrpE-like protein; the predicted start codon of grpE overlaps the last two codons of dnaK, indicating that the two genes are translationally coupled. This organisation differs from that reported for other prokaryotes.

A New Method to Value Efficiency of Enzyme Blends for Pancreatic Tissue Digestion

Islet transplantation, since the 1990s, has been an example of human cell therapy. Nevertheless, the islet isolation procedure is not completely standardized; in fact, >50% of islet procedures do not eventuate in transplantation due both to the variability of a donors pancreas and to the unpredictable efficiency of an enzymatic blend. The enzymes used in pancreas isolation to digest several substrates are extracted from Clostridium histolyticum. In particular, they have strong collagenolytic activity compared with vertebrate collagenases. However, several impediments persist in human islet isolation success, probably owing to the variable composition and concentration of collagenases employed during the digestion phase. For islet isolation processes, neutral proteases play important roles. However, they should be considered to be double-edged swords, contributing to tissue dissociation but, sometimes, decreasing islet yield through fragmentation, breakdown, and inactivation. Protease activities cannot be preciously adjusted in a narrow range, there is no approach to determine the optimal dosage and composition of enzymes for extraction of human islets from the pancreas. At this time, available data on commercial enzymatic activity are not sufficient to predict their efficiency for pancreas digestion; consequently, it is difficult to select enzyme batches. For these reasons, we sought to generate an innovative evaluation assay to select enzymes useful for isolation procedures of pancreatic islets.

Role of S128R polymorphism of E-selectin in colon metastasis formation

The extravasation of cancer cells is a key step of the metastatic cascade. Polymorphisms in genes encoding adhesion molecules can facilitate metastasis by increasing the strength of interaction between tumor and endothelial cells as well as impacting other properties of cancer cells. We investigated the Ser128Arg (a561c at the nucleotide level) polymorphism in the E‐selectin gene in patients with metastatic colon cancer and its functional significance. Genotyping for a561c polymorphism was performed on 172 cancer patients and on an age‐matched control population. The colon cancer group was divided into groups with (M+) and without observable metastasis (M−). For in vitro functional assays, Huvec transfected cells expressing wild‐type (WT) or the S128R variant of E‐selectin were established to study in vitro binding ability and signal transduction processes of T84 colon cancer cell line. Our results demonstrated that the Arginine128 allele was more prevalent in the M+ group than in the M− group or normal controls (p < 0.005; odds ratio, 1.56; 95% confidence interval (CI) 1.16–1.92; p < 0.001, odds ratio = 1.65; CI = 1.24–1.99, respectively). In vitro, S128R E‐selectin transfected Huvec cells, supported increased adhesion as well as increased cellular signaling of T84 cancer cells compared to WT E‐selectin and mock‐transfected Huvec cells. These findings suggest that the E‐selectin S128R polymorphism can functionally affect tumor‐endothelial interactions as well as motility and signaling properties of neoplastic cells that may modulate the metastatic phenotype.

Screening of subtelomeric rearrangements in autistic disorder: Identification of a partial trisomy of 13q34 in a patient bearing a 13q;21p translocation

Within the framework of a FISH screening protocol to detect cryptic subtelomeric rearrangements in autistic disorder (AD), a patient bearing three copies of the subtelomeric portion of the q arm of chromosome 13 has been identified. Beside AD, the patient also has severe mental retardation and displays several dysmorphic features. Further FISH analyses revealed that the trisomy was caused by the translocation of a 13q subtelomeric fragment to the acrocentric tip of one chromosome 21 [46,XY.ish der(21) t(13;21) (q34;p13)(D13S1825+)]. Gene dosage experiments carried out with three multiallelic polymorphisms of the subtelomeric region of chromosome 13q showed that the putative length of the triplicate region does not exceed 300 kb about, that is, the distance from telomere to the first normally inherited marker. In addition, gene dosage analysis performed on the derivative chromosome 21, did not reveal loss of the most telomeric protein‐encoding genes on 21p. The potential relationship between a postulated increased expression of genes on 13q34 and the complex phenotype in this trisomic patient is discussed.

Analysis of the gastrin-releasing peptide receptor gene in Italian patients with autism spectrum disorders†

The gastrin‐releasing peptide receptor (GRPR) was implicated for the first time in the pathogenesis of Autism spectrum disorders (ASD) by Ishikawa‐Brush et al. [Ishikawa‐Brush et al. (1997): Hum Mol Genet 6: 1241–1250]. Since this original observation, only one association study [Marui et al. (2004): Brain Dev 26: 5–7] has further investigated, though unsuccessfully, the involvement of the GRPR gene in ASD. With the aim of contributing further information to this topic we have sequenced the entire coding region and the intron/exon junctions of the GRPR gene in 149 Italian autistic patients. The results of this study led to the identification of four novel point mutations, two of which, that is, C6S and L181F, involve amino acid changes identified in two patients with ASD and Rett syndrome, respectively. Both the leucine at position 181 and the cysteine at position 6 are strongly conserved in vertebrates. C6S and L181F mutant proteins were expressed in COS‐7 and BALB/3T3 cells, but they did not affect either GRPs binding affinity or its potency for stimulating phospholipase C‐mediated production of inositol 1,4,5‐trisphosphate. In summary, our results do not provide support for a major role of the GRPR gene in ASD in the population of patients we have studied. However, there is a potential role of C6S and L181F mutations on GRPR function, and possibly in the pathogenesis of the autistic disorders in the two patients.

Suggestive evidence for association of D2S2188 marker (2q31.1) with autism in 143 Sicilian (Italian) TRIO families.

We have screened 143 Sicilian (Italian) families with one autistic child to verify, by a linkage disequilibrium approach, the involvement of the 2q31.1 region in the cause of the disease in these families. Our study design includes the use of intrafamilial association to prevent a population stratification bias and ethnic homogeneity of the sample. The results of our analysis provided suggestive evidence of the occurrence of transmission disequilibrium between autism and the D2S2188 polymorphism in Sicilian TRIO families, a finding which provides further and independent support to the hypothesis of the existence of a susceptibility gene (or genes) for autism on chromosome 2q.