Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice.

The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.

Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex.

The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.

Application of the reaction of dithioesters with epsilon-amino groups in lysine to the chemical modification of proteins.

The reaction of lysine with dithioesters was applied to horseradish peroxidase donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) using carboxymethyl dithiotridecanoate: three to four lysine residues were modified. The modified enzyme was soluble and active in diethyl ether. Papain (EC 3.4.22.2) was modified with carboxymethyl dithiobenzoate: two lysine residues were modified. The modified enzyme was soluble and active in dimethylsulfoxide. From these results it is concluded that dithioesters are efficient reagents for the modification of peripheral lysine residues of proteins. Aromatic dithioesters, less reactive but more selective, should be recommended for thiol-dependent enzymes such as papain.

Hyperinsulinemia and abdominal obesity affect the expression of hypertriglyceridemia in heterozygous familial lipoprotein lipase deficiency.

We have reported three missense mutations (G188E, P207L, and D250N) in the lipoprotein lipase (LPL) gene among French-Canadians, resulting in the absence of measurable postheparin plasma LPL activity in homozygotes. Presence of triglyceride- and cholesterol-rich VLDL, as well as cholesterol-poor HDL particles, has been shown in heterozygotes affected by partial reduction in postheparin LPL activity. However, significant heterogeneity in their plasma triglyceride levels has been found, even among individuals carrying the same LPL gene mutation, indicating that factors other than LPL deficiency could affect the phenotypic expression of hypertriglyceridemia in the heterozygous state. The aim of the present study was to examine the combined effects of abdominal fat accumulation and hyperinsulinemia on plasma triglyceride levels among heterozygous patients for familial LPL deficiency. Based on sex and BMI, 43 heterozygotes (25 women and 18 men) were matched with noncarrier control subjects. Our data indicate that heterozygotes with higher abdominal fat deposition, as defined as waist girth values above the 50th percentile, had higher plasma triglyceride levels than nonobese heterozygotes. However, an important proportion of male heterozygote subjects were hypertriglyceridemic, even in absence of abdominal obesity, suggesting that another factor(s) was involved in the modulation of hypertriglyceridemia in these subjects. Indeed, multivariate analyses revealed that fasting hyperinsulinemia was a significant correlate of hypertriglyceridemia among these heterozygotes. Results of the present study indicate that abdominal obesity and hyperinsulinemia both have deleterious effects on plasma triglyceride levels in familial LPL deficiency. It is suggested that heterozygotes with moderate obesity and/or insulin resistance may be at higher risk of coronary artery disease because of the expression of an atherogenic lipoprotein phenotype among these patients.

Normal brain development in PS1 hypomorphic mice with markedly reduced gamma-secretase cleavage of betaAPP.

Presenilin 1-null mice die at birth from brain and skeletal developmental deformities due to disrupted Notch signaling. Presenilin 1-null mice also have severely reduced gamma-secretase cleavage of betaAPP. The assumption has been that facilitation of Notch signaling and betaAPP processing by presenilin 1 are analogous functions. Here we describe a presenilin 1-targetted mouse model that expresses extremely low levels ( approximately 1% of normal) of mutant PS1-M146L. Homozygous mice have significantly reduced viability due to a Notch-like phenotype. The animals that survive have severe axial skeletal deformities and markedly diminished gamma-secretase activity and accumulation of betaAPP-C100, but no obvious abnormalities in brain development. These results suggest that, in mice, a marked reduction of PS1-facilitated gamma-secretase activity is not detrimental to normal brain development.

Presenilin 1 is actively degraded by the 26S proteasome.

The metabolic pathways governing the turnover of presenilin 1 (PS1) have been incompletely worked out. The PS1 holoprotein has low abundance in many cells and appears to undergo endoproteolytic cleavage near residue 298. We provide evidence that one mechanism by which the PS1 holoprotein is degraded is through the action of the 26S proteasome. We also show that the proteasome does not participate in the endoproteolytic cleavage.

Determination of changes in specific gene expression by reverse transcription PCR using interspecies mRNAs as internal standards.

A method is described for the determination of changes in gene expression by reverse transcription of the target mRNA followed by PCR amplification of the resulting cDNA (RT-PCR), using the lipoprotein lipase gene as the model system. Known proportions of human and rat adipose tissue homogenates are mixed and are processed together throughout the RT-PCR procedure so that the rat tissue serves as an internal standard for the measurement of human adipose tissue lipoprotein lipase (LPL) in all steps including RNA extraction, reverse transcription and PCR amplification. Taking advantage of the highly conserved sequence of the LPL gene across species, selected homologous regions of the human and rat genes are amplified using the same primer pair and resulting in the same lengths of amplified DNA fragments. The two amplified products are then separated from each other by making use of differences in the position of a restriction site in the two amplified DNA fragments. The method is simple, precise and reproducible and avoids construction of tailored nucleic acids for use as internal standards.

Amyloid-beta-protein isoforms in brain of subjects with PS1-linked, beta APP-linked and sporadic Alzheimer disease.

To determine whether similar abnormalities of various soluble full-length and N-terminal truncated Abeta peptides occur in postmortem cerebral cortex of affected PS1 mutation carriers, we examined the amounts of two amyloid species ending at residue 40 or at residues 42(43) using sandwich ELISA systems. Our results indicate that PS1 mutations effect a dramatic accumulation in brain of the highly insoluble potentially neurotoxic long-tailed isoforms of the Abeta peptide such as Abeta1-42(43) and Abetax-42(43). This enhancing effect of PS1 mutation on Abetax-42(43) deposition was highly similar to that of a betaAPP mutation (Val717Ile) but the effects on Abetax-40 production were significantly different between these two causal genes. In contrast to previous studies of soluble Abeta in plasma and in supernatants from cultured fibroblasts of subjects with PS1 mutations, our studies also show that there is an increase in insoluble Abetax-40 peptides in brain of subjects with PS1 mutations.

Isolation of free and membrane-bound polysomes and mRNA highly active in translation and reverse transcription from small discrete regions of rat brain.

A procedure is described for the preparation of total polysomes, membrane-bound and free polysomes and polysomal mRNA from as little as 5 mg or less of brain tissue. These preparations were highly active when tested for translation and reverse transcription in vitro. Using this method, polysomes and mRNA from rat cerebral cortex, cerebellum, hippocampus and hypothalamus were compared. The results showed that membrane-bound polysomes were more active than free polysomes in protein synthesis. The activities of polysomes and mRNA for protein and cDNA synthesis were dependent on the specific brain structures from which they were obtained. Polysomes from cerebellum and hypothalamus incorporated amino acids more actively than those from cerebral cortex or hippocampus, when tested in a reticulocyte lysate system. Cerebellar mRNA also showed the highest activity for cDNA syntehsis as compared to mRNAs from the other three tissues.

Possible involvement of the amygdaloid complex in morphine analgesia as studied by electrolytic lesions in rats.

The analgesic effects of morphine (5 mg/kg i.p.) were studied in biamygalectomized rats. (1) Using the tail-flick test neither withdrawal latencies nor morphine time-course and efficacy were affected by the lesions. (2) The threshold for vocalization to electrical stimulation of the tail was greatly increased in lesioned rats; however, statistical analysis revealed no significant change in the analgesic efficacy of morphine.

Failure to detect missense mutations in the S182 gene in a series of late-onset Alzheimer's disease cases.

The possibility of an interaction of multiple genes has been speculated in pathogenesis of Alzheimer's disease (AD). Because we have recently cloned a novel gene S182 bearing five different missense mutations which segregate with early-onset familial AD, we sought the frequency of these mutations in familial and sporadic late-onset AD to clarify the incidence of these mutations in the disease. The current study showed lack of these mutations in 118 independent subjects affected with late-onset Alzheimer's disease.

Absence of association between Alzheimer disease and the -491 regulatory region polymorphism of APOE.

A novel polymorphism (-491 A/T) within the regulatory region on the apolipoprotein E gene has recently been reported to be associated with risk for Alzheimer disease (AD). To test this association in an independent data set, we have examined this polymorphism in a sample of 88 well-characterized AD cases and compared the allele frequency and genotype frequencies for this polymorphism with those observed in 112 cognitively normal subjects drawn from the same ethnic group. These results suggest that in the current data set at least, the -491 A/T polymorphism is not associated with risk for AD, but may be in partial linkage disequilibrium with the APOE epsilon2/epsilon3/epsilon4 polymorphism.

Effects of trichothecenes (T-2 toxin) on protein synthesis in vitro by brain polysomes and messenger RNA.

The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate in vitro systems pretreated with micrococcal nuclease. One of the test systems contained purified globin mRNA and was initiation dependent. The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process. T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10(-8) M to 10(-4) M. Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA. While high concentrations of the toxin (10(-4) M) led to partial inhibition of protein synthesis by polysomes, low concentrations (10(-8) M and 10(-6) M) stimulated protein synthesis. Comparison of the above results with those obtained by other workers suggest that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system. A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.

Sexual differences in dental development and prediction of emergence.

The dental development of a genetically homogeneous French-Canadian group of children ranging in age from 2.5 to 19 years was evaluated from 5,437 panoramic radiographs by the method of Demirjian et al. The maturity of each mandibular tooth was evaluated individually. For each stage of each tooth, the developmental curves of boys and girls were compared. A common pattern was found for each tooth, namely the chronological similarity between boys and girls in the early stages of development and the advancement of girls over boys for the later stages. Development scores were given to the seven mandibular teeth. Up to five to six years of age, no difference was found in the timing of dental development between boys and girls, in contrast to the older ages where girls were always more developed than boys. When the emergence curve was plotted with developmental curves, a close relation was established between the stage of formation of all teeth and their emergence, hence the predictive value of the use of dental maturity curves in clinics.

The inter-examiner variation in rating dental formation from radiographs.

Six examiners evaluated dental formation on 191 radiographs, using an eight-stage system. Discrepancies between these examiners are generally of the order of +/- one stage, in about 20 to 25% of the cases. The authors thus suggest the use of reference radiographs, for the double purpose of ensuring a homogeneous rating by two examiners or more, as well as the assessment of eventual differences between populations.

Sexual dimorphism in the development, emergence, and agenesis of the mandibular third molar.

Bilateral agenesis was encountered in about 9% of the cases, with no significant sexual difference. The right and left mandibular third molars had the same pattern of development and emergence. The slight advance of girls over boys at the crown-completion stage was similar to previous observations on other mandibular teeth, particularly the second molar. The root development course of the third molar was faster in males than in females; this sexual dimorphism was much greater for retarded cases than for advanced cases. At the apex closure, the difference between median ages of males and females was 1.5 yr. Alveolar emergence tended to occur at a lower developmental stage in advanced cases compared with retarded cases.

Epileptic discharges induced by intermittent light stimulation in photosensitive baboons: a current source density study.

The current source density (CSD) method was applied to the study of paroxysmal discharges (PDs) induced by intermittent light stimulation (ILS) in Papio papio baboons made photosensitive by a subconvulsant dose of allylglycine. CSD was studied in the motor and premotor areas (4 and 6). Laminar profiles of sinks and sources are similar in both areas. Nevertheless, the motor area seems to become involved first since it shows the earliest and most prominent sink in layer III. Such a sink, correlated with the PD spike, moves progressively upward to the cortical surface. The localization and other experimental arguments obtained by the same method suggest that this sink could be mainly of dendritic origin. The cortico-cortical afferents to the superficial layers of the motor area might thus determine the generation of this sink. A smaller sink, detected at the same latency between layers V and VI could correspond to synaptic activations due to thalamo-cortical afferents probably arriving on the pyramidal cells which project to the spinal cord. Intense sinks correlated with the PD wave in layer V could be passive, due to active sources lying just above and/or below, because in previous studies an inhibition of the cellular discharges was always observed in correlation with the wave. It is suggested that ILS triggered PDs involve visual cortico-cortical afferents directed mainly to the superficial layers of the motor area provoking an intense synaptic activation of the cellular elements situated at this level.

Survey of stalking at WPI.

The purpose of this study was to ascertain the amount of stalking taking place on the Worcester Polytechnic Institute (WPI) campus in which computers play such a huge role in the lives of the students. Our results were then compared with those reported in a stalking study done at West Virginia University (WVU). Surprisingly (to us), a smaller percentage of both females and males were stalked at WPI. The use of the Internet did not play a major role in stalking as we had expected. Results reported in a TV news report and a newspaper article indicate, however, that much less stalking occurs among the general population than does at WVU and WPI.

[Long acting sandostatine (sandostatine LAR) in the treatment of acromegaly]. / La sandostatine retard (sandostatine LAR) dans le traitement de l'acromégalie.

A long-acting depot formulation of octreotide (Sandostatin LAR, Sandoz LTD) has been recently developed. Preliminary studies indicated that, in acromegalic patients previously controlled by Sandostatin 300-600 micrograms/day in 2-3 sc injections, the intramuscular administration of 20-30 mg of Sandostatin LAR achieved, during one month a similar control of GH hypersecretion. In the present study, the variations of plasma levels of octreotide, GH and IGF1 were followed during 2 months in acromegalic patients receiving a unique injection of 20 mg (n = 4) or 30 mg (n = 4) of Sandostatin LAR. Following Sandostatin LAR 20 mg i.m, the baseline values of GH (8.1 +/- 2.5 micrograms/l) and IGF1 (684 +/- 92 micrograms/l) were normalized after 2 weeks and remained into the normal range during the 28 following days. Similar results were obtained, after a 30 mg i.m administration of Sandostatin LAR. In this later case, the maximal inhibition of GH and IGF1 (1.3 +/- 1.0 micrograms/l and 392 +/- 266 micrograms/l respectively) lasted 2 months. These data showed that a monthly injection of Sandostatin LAR (20-30 mg) allowed a correct control of GH hypersecretion in this series of acromegalic patients.

[Lp(a) and weight loss in obese patients]. / Lp(a) et perte de poids chez l'obèse.

High concentrations of lipoprotein (a) (Lp(a)) are associated with an increased risk of atherosclerotic vascular disease. Lp(a) synthesis is mainly under genetic control but many endocrine disturbances may modulate Lp(a) plasmatic concentrations. There is no agreement upon Lp(a) variations in patients under a hypocaloric diet. This study was undertaken to assess this point in obese females subjected to a 1100 kcal/d diet. Ninety-two obese patients (42.4 +/- 10.4 yr old, BMI 33.9 +/- 5.6 kg/m2) came once a week as out patients during 9 weeks. Lp(a) concentrations distribution was highly skewed. The threshold Lp(a) concentration for a significant cardiovascular risk is estimated at 0.3 g/l. Concentrations above 0.3 g/l were found in 29/92 patients (31%). If the patients were distributed in 2 groups according to their Lp(a) values (< or = ou > 0.3 g/l), the BMI, total cholesterol or triglycerides were not different. There were no significant correlation between Lp(a) and age, total cholesterol or triglycerides. After 9 weeks BMI and total cholesterol values decreased (-1.6 +/- 3.4 kg/m2 and -0.17 +/- 0.68 mmol/l, respectively). Lp(a) concentrations were unchanged (0.3 +/- 0.3 vs 0.3 +/- 0.3 g/l). There were no significant correlation between Lp(a) variations and age, BMI or initial Lp(a) concentrations. No significant decrease of Lp(a) could be detected even in the sub-group of patients with initial concentrations of Lp(a) > 0.3 g/l or even in a sub-group with Lp(a) > 0.7 g/l (n = 7). Under our conditions, weight loss is not associated with a decrease of Lp(a) concentrations suggesting that in a given obese a single determination is enough to assess his Lp(a)-related atherosclerotic risk.