Rosa Adroer

Evolution of protamine P1 genes in mammals

Prolamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that prolamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5–6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically.

Evolution of protamine P1 genes in primates

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10–13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24–29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39–44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved.

Apolipoprotein E4 allele frequency in Spanish Alzheimer and control cases

We have found an APOE epsilon 4 allelic frequency of 0.289 (95% CI 0.195-0.383) in Spanish AD patients (n = 88; average age = 71.2 +/- 9.37) and of 0.061 (95% CI 0.023-0.099) in age-matched controls (n = 147; average age = 71.5 +/- 10.29). Remarkably no ApoE 4/4 subjects were observed in any of the age-matched control groups compared to a total of 22 AD patients with the ApoE 4/4 phenotype. The combined odds ratio for subjects with one or two epsilon 4 alleles in the present study is 6.25 (95% CI 3.13-12.60), which is one of the highest so far reported. Altogether our results suggest a trans-European difference in the ApoE epsilon 4 frequency but no differences in the strength of the association between APOE4 and AD.

Calcium precipitation in acute and chronic brain diseases.

In rat brain, calcification associated with excitotoxicity has been proposed to play a protective role, whereas in human brain, nonartherosclerotic calcification is present in several pathological conditions without any clear significance. To determine if calcification can be viewed as a protective step of calcium homeostasis during chronic and acute neuronal suffering, cerebral cortex and hippocampus of patients with Alzheimers disease, vascular dementia and neonatal hypoxia-ischemia were investigated. To investigate the human specificity, these two areas were also studied in dogs with established cognitive deficits. In all groups, calcium precipitates were observed in the cerebral parenchyma associated with neuronal damage. The cerebral cortex presented a higher degree of calcification than the hippocampus. The neonatal hypoxia-ischemia group was characterised by a higher degree of calcification, whereas the groups with lowest calcification were the Alzheimers patients and dogs. As shown by X-ray microanalysis, in the precipitates, calcium is mainly associated with phosphorus in a form that resembles hydroxyapatites. Thus, intracellular calcium concentration associated with neuronal suffering may reduce the energy extrusion. We propose that, to help overcome excitotoxicity, calcium precipitation acts in CNS of vertebrates as a new compartment of the calcium homeostasis in which free cytoplasmic calcium ions are inactivated by phosphate ones.

Apolipoprotein E Polymorphism in Alzheimer’s Disease: A Comparative Study of Two Research Populations from Spain and the United States

We examined the distribution of the apolipoprotein E (APOE) polymorphism in two Caucasian populations of Alzheimer’s disease (AD) patients referred to dementia clinics; one in Gerona, Spain (66 AD patients, 49 controls), and the other in Pittsburgh, Pa., USA (209 AD patients, 58 controls). The presence of the APOE*4 allele was a significant risk for developing AD in both cohorts: Gerona (odds ratio = 2.34, CI: 1.03–5.55) and Pittsburgh (odds ratio = 3.64, CI: 1.78–7.69). The proportion of AD with the APOE*4 allele was greater in the Pittsburgh cohort than in the Gerona cohort (p = 0.02). However, no statistical difference was noted between the two populations in nondemented controls (p = 0.41). These data emphasize the importance of geographical and ethnic variations in the study of APOE genotypes.

Conserved elements in the 5' regulatory region of the amyloid precursor protein gene in primates.

Oligonucleotides corresponding to conserved sites between the human and mouse amyloid precursor protein (APP) genes have been used to polymerase chain reaction (PCR) amplify and sequence the promoter region of the APP gene from chimpanzee, gorilla, orang-utan, papio and African green monkey. Several novel conserved potentially-regulatory sequences of the APP gene have been detected after alignment of the APP promoter sequences: an apolipoprotein E-B1 (APOE-B1) element at position -450, also present in the APOE gene, two activator protein-2 (AP-2) sites at positions -450 and -301 and an intermediate early-1 gene (IE1) site at position -280. These elements are conserved in all mammalian APP promoter sequences studied. Additionally a previously detected heat shock element (HSE) at position -317, and an activator protein-1 (AP-1) site at position -350 are also conserved. Knowledge of the essential regulatory elements at the APP gene constitute the basis for understanding its transcriptional control and subsequent model studies.

A novel silent variant at codon 711 and a variant at codon 708 of the APP sequence detected in Spanish Alzheimer and control cases

Pathogenic mutations have been identified in exons 16 and 17 of the beta-amyloid precursor protein (APP) gene in some cases of early onset Alzheimers disease. Screening of these exons in a number of familial and sporadic cases of Alzheimers disease in Spain, resulted in the identification of a novel silent variant at codon 711 whose relevance to the AD pathogenesis remains unclear. The 708 variant was also detected in one of normal controls.

Improved direct sequencing of Alzheimer's amyloid precursor protein (APP) exons 16 and 17

Direct sequencing of exon 17 of the amyloid precursor protein (APP) gene led to the identification of 3 different types of APP717 pathogenic mutations associated with familial Alzheimers disease (FAD). The low frequency of these mutations results in having to screen many samples in order to identify new families affected by them, which is laborious and time consuming. Thus, in order to help the identification of these mutations in additional countries and to search for new mutations in APP, perhaps in other exons also causing FAD, we have optimized the procedure and reduced the time necessary for sample preparation from 11 h to 3 1/2 h.

Direct Sequencing of the Human Protamine P1 Gene and Application in Forensic Medicine

Protamines are among the most variable nuclear proteins known in eukaryotes. In order to learn more about their evolution and function in humans and to explore the possibility of potential applications in forensic medicine we have developed a rapid method to amplify and directly sequence the protamine P1 gene simultaneously in many different samples. The method takes only 3.5 h from genomic DNA to the sequencing reactions. Despite the high variability of these genes only one polymorphic site was detected at the coding region level in different individuals. This polymorphic variation does not create a change in the amino-acid sequence of the protamine. Because all the protamine genes sequenced from different species are markedly different among them as well as to the human sequence, amplification and direct sequencing of this gene can be used to unequivocally identify the human or animal origin of biological specimens. Furthermore, the single polymorphic site detected in the human P1 gene could be useful in conjunction with other markers in identification studies in humans.

Low apolipoprotein E ε4 allele frequency in the population of Catalonia (spain) determined by Pcr-Rflp and Laser fluorescent sequencer

Specific apolipoprotein E alleles have been associated in the last few years with several diseases using appropriate controls. However, these control groups are rarely representative of the general population since they correspond either to aged or healthy control groups (and thus depleted of pathological alleles). For this reason it is difficult at present to compare population allelic frequencies in different countries. In order to provide this essential basic data representative of the general population, in this work we have determined the distribution of apolipoprotein E alleles in 226 individuals from the population of Catalonia (Spain) sampled with the main purpose of paternity testing. The allelic frequencies are: ε2 = 0.064, ε3 = 0.810 and ε4 = 0.126, predicting a lower incidence of Alzheimer disease and possibly also of other pathologies where this allele is a risk factor.