Structure activity relationship towards design of cryptosporidium specific thymidylate synthase inhibitors.

Cryptosporidiosis is a human gastrointestinal disease caused by protozoans of the genus Cryptosporidium, which can be fatal in immunocompromised individuals. The essential enzyme, thymidylate synthase (TS), is responsible for de novo synthesis of deoxythymidine monophosphate. The TS active site is relatively conserved between Cryptosporidium and human enzymes. In previous work, we identified compound 1, (2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidin-methyl-phenyl-l-glutamic acid), as a promising selective Cryptosporidium hominis TS (ChTS) inhibitor. In the present study, we explore the structure-activity relationship around 1 glutamate moiety by synthesizing and biochemically evaluating the inhibitory activity of analogues against ChTS and human TS (hTS). X-Ray crystal structures were obtained for compounds bound to both ChTS and hTS. We establish the importance of the 2-phenylacetic acid moiety methylene linker in optimally positioning compounds 23, 24, and 25 within the active site. Moreover, through the comparison of structural data for 5, 14, 15, and 23 bound in both ChTS and hTS identified that active site rigidity is a driving force in determining inhibitor selectivity.

Metals in plasma of nonagenarians and centenarians living in a key area of longevity.

The concentration of calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn) and selenium (Se) in plasma of 76 nonagenarians (mean age, 89.0±6.3 years), 64 centenarians (mean age, 101±1 years) and 24 middle-aged subjects as controls (mean age 61.2±1.1 years), was determined by sector field inductively coupled plasma mass spectrometry. All the subjects lived in Sardinia, an Italian island, that has the higher prevalence of centenarians than in other European countries. A comparison among the three classes of age showed a significant depletion of Ca, Co, Fe, Mn and Se (all p<0.001) in nonagenarians and centenarians with respect to controls. In particular, the geometric mean (GM) values of Ca, Co, Fe, Mn and Se were: 94.1 µg/ml, 0.46 ng/ml, 1314 ng/ml, 2.47 ng/ml and 111 ng/ml in controls; 87.6 µg/ml, 0.22 ng/ml, 815 ng/ml, 1.07 ng/ml and 88.9 ng/ml in nonagenarians; 87.0 µg/ml, 0.29 ng/ml, 713 ng/ml, 1.27 ng/ml and 81.9 ng/ml in centenarians. The highest inverse relationship with age was observed for Fe (p<0.001; ρ=-0.352) and Se (p<0.001; ρ=-0.417). This trend was also observed when data were sorted by gender. On the other hand, Cu and Mg levels in plasma remained substantially unchanged during aging. As regards Cu, it was significantly higher in females than in males in controls (GM, 1294 ng/ml vs. 1077 ng/ml; p=0.012), in nonagenarians (GM, 1216 ng/ml vs. 1081 ng/ml; p=0.011) as well as in centenarians (GM, 1226 ng/ml vs. 1152 ng/ml; p=0.045) and in hypertensive subjects with respect to healthy people (GM, 1215 ng/ml vs. 1129 ng/ml; p=0.021). These data can be used to enhance knowledge and support the research on: i) metals involved in aging in areas with high rates of human longevity; ii) variables (gender, lifestyle habits and health status) as critical determinants in aging; and iii) mineral intake and supplementation at older age affecting the healthy aging.

Design, recruitment, logistics, and data management of the GEHA (Genetics of Healthy Ageing) project.

In 2004, the integrated European project GEHA (Genetics of Healthy Ageing) was initiated with the aim of identifying genes involved in healthy ageing and longevity. The first step in the project was the recruitment of more than 2500 pairs of siblings aged 90 years or more together with one younger control person from 15 areas in 11 European countries through a coordinated and standardised effort. A biological sample, preferably a blood sample, was collected from each participant, and basic physical and cognitive measures were obtained together with information about health, life style, and family composition. From 2004 to 2008 a total of 2535 families comprising 5319 nonagenarian siblings were identified and included in the project. In addition, 2548 younger control persons aged 50-75 years were recruited. A total of 2249 complete trios with blood samples from at least two old siblings and the younger control were formed and are available for genetic analyses (e.g. linkage studies and genome-wide association studies). Mortality follow-up improves the possibility of identifying families with the most extreme longevity phenotypes. With a mean follow-up time of 3.7 years the number of families with all participating siblings aged 95 years or more has increased by a factor of 5 to 750 families compared to when interviews were conducted. Thus, the GEHA project represents a unique source in the search for genes related to healthy ageing and longevity.

High-throughput capillary electrophoresis method for plasma cysteinylglycine measurement: evidences for a clinical application.

Increased levels in plasma homocysteine and cysteine, and more recently, decreased levels in cysteinylglycine have been indicated as a risk factor for vascular diseases. Most assays focused their attention only on homocysteine determination and when also other thiols were measured, analytical times drastically increased. By modifying our previous method for thiols detection, we set up a rapid capillary electrophoresis method for the selective quantification of plasma cysteinylglycine, cutting the analysis time of about 50%. Samples were treated with tri-n-butylphosphine as reducing agent, proteins were precipitated with trichloroacetic acid and released thiols were successively derivatized by the selective thiol laser-induced fluorescence-labeling agent 5-iodoacetamidofluorescein and separated by capillary electrophoresis. A baseline separation between peaks was obtained in about 2 min using 3 mmol/L sodium phosphate/2.5 mmol/L boric acid as electrolyte solution with 75 mmol/L N-methyl-D-glucamine at pH 11.25 in a 47 cm long capillary with a cartridge temperature of 45 degrees C. The method application was checked by measuring plasma Cys-Gly levels in a group of patients affected by retinal vein occlusion (RVO), an important cause of visual loss in the elderly. The low levels of Cys-Gly found in the RVO patients suggest that these small thiols may have importance in the disease development.

Effect of acute exercise on low molecular weight thiols in plasma.

An important defence against free radicals is represented by plasma low molecular weight (LMW) thiols that compose a dynamic system of reduced and oxidized forms able to act as a buffer redox system. This study examined the effect of an acute graded exercise bout on LMW thiols in 16 young subjects (six sedentaries and 10 athletes). Blood analysis was performed before and immediately after the exercise and total and reduced thiols were measured in order to evaluate the thiol redox status. Findings suggested that the exercise test proposed was not enough to imbalance the redox status of all LMW thiols. However, when the redox status was evaluated for each thiol, it was evident that homocysteine (Hcy) redox status was significantly different after physical activity. In particular, we found a lower level of reduced Hcy after the exercise test both in sedentaries and in athletes. We concluded that duration and intensity of the proposed exercise were not enough to promote a reactive oxygen species production able to imbalance the redox thiols status and that the lowering of the reduced Hcy form may be due to the effect produced during the effort on the synthesis and/or removal processes of Hcy.

Different antioxidant profiles in Italian centenarians: the Sardinian peculiarity.

In this study, 153 Italian centenarians from four different geographical areas, including Modena (northern Italy), Ancona (central Italy), Perugia (central Italy) and Sardinia island (AKEA Project) were enrolled. Plasma levels of vitamin C, uric acid, vitamin A and vitamin E as well as the activities of superoxide dismutase and glutathione peroxidase were measured. Subjects were compared to a younger control population of the same areas, divided into three age groups:

Brain perfusion abnormalities in patients with euthyroid autoimmune thyroiditis.

PURPOSE: Brain perfusion abnormalities have recently been demonstrated by single-photon emission computed tomography (SPECT) in rare cases of severe Hashimoto's thyroiditis (HT) encephalopathy; moreover, some degree of subtle central nervous system (CNS) involvement has been hypothesised in HT, but no direct evidence has been provided so far. The aim of this study was to assess cortical brain perfusion in patients with euthyroid HT without any clinical evidence of CNS involvement by means of 99mTc-ECD brain SPECT. Sixteen adult patients with HT entered this study following informed consent. METHODS: The diagnosis was based on the coexistence of high titres of anti-thyroid auto-antibodies and diffuse hypoechogenicity of the thyroid on ultrasound in association with normal circulating thyroid hormone and TSH concentrations. Nine consecutive adult patients with non-toxic nodular goitre (NTNG) and ten healthy subjects matched for age and sex were included as control groups. All patients underwent 99mTc-ECD brain SPECT. Image assessment was both qualitative and semiquantitative. Semiquantitative analysis was performed by generation of four regions of interest (ROI) for each cerebral hemisphere--frontal, temporal, parietal and occipital--and one for each cerebellar hemisphere in order to evaluate cortical perfusion asymmetry. The Asymmetry Index (AI) was calculated to provide a measurement of both magnitude and direction of perfusion asymmetry. RESULTS: As assessed by visual examination, 99mTc-ECD cerebral distribution was irregular and patchy in HT patients, hypoperfusion being more frequently found in frontal lobes. AI revealed abnormalities in 12/16 HT patients, in three of the nine NTNG patients and in none of the normal controls. A significant difference in the mean AI was found between patients with HT and both patients with NTNG (p<0.003) and normal controls (p<0.001), when only frontal lobes were considered. CONCLUSION: These results show the high prevalence of brain perfusion abnormalities in euthyroid HT. These abnormalities are similar to those observed in cases of severe Hashimoto's encephalopathy and may suggest a higher than expected involvement of CNS in thyroid autoimmune disease.

Y chromosome binary markers to study the high prevalence of males in Sardinian centenarians and the genetic structure of the Sardinian population.

We have analyzed a sample of 40 centenarians and 116 young controls from Sardinia, with a set of new Y chromosome binary markers, to evaluate if Y chromosome genes are involved in the high prevalence of males among centenarian Sardinians (1/2 vs. 1/4 in other populations studied). The results indicate that none of the seven lineages that account for >97% of the Y chromosome diversity in Sardinia provide an advantage with respect to the extreme longevity. However, our results, although based on the male-specific Y chromosome polymorphisms, give a clear profile of the pattern of genetic variability in Sardinia. Indeed they indicate that the Sardinian population had two main founder populations that have evolved in isolation for at least the last 5,000 years. These findings set the stage for future studies on longevity and other complex traits in Sardinia.

Long-term dehydroepiandrosterone and 16alpha-fluoro-5-androsten-17-one administration enhances DNA synthesis and induces expression of c-fos and c-Ha-ras in a selected population of preneoplastic lesions in liver of diethylnitrosamine-initiated rats.

Dehydroepiandrosterone (DHEA) inhibits glucose 6-phosphate dehydrogenase (G6PD) activity and growth of preneoplastic lesions in various tissues, but its administration may also enhance tumorigenesis by genotoxic carcinogens. We have investigated in single preneoplastic liver lesions, induced in diethylnitrosamine-initiated rats by the resistant hepatocyte protocol, the mechanisms underlying these opposite DHEA effects. Administration of DHEA (0.45% in the diet) for 10 and 26 weeks and of its analog 16alpha-fluoro-5-androsten-17-one (FA, 0.25%) for 10 weeks, starting 4 weeks after initiation, induced an apparent decrease in the number of glutathione S:-transferase (placental) (GST-P)-positive lesions and an increase in lesion volume. DHEA administration for 38 weeks enhanced hepatocellular carcinoma multiplicity. Depending on the rise in the number of slowly growing, remodeling GST-P-positive lesions induced by DHEA and FA, overall DNA synthesis decreased slightly in these lesions at 14 weeks, but increased in uniform lesions. Labeling index (LI) in single uniform lesions at 14 weeks ranged between very low (not different from normal liver) to high (>10-fold normal liver). DHEA and FA induced broad increases in lesions with a high LI, which showed a higher number of cells overexpressing c-Ha-ras and/or c-fos than those with a lower LI. High G6PD activity was inhibited by DHEA and FA in only approximately 50% of preneoplastic lesions. These data indicate selection in rats subjected to long-term DHEA and FA treatments of a subpopulation of GST-P-positive cells with high growth and progression potentials. Overall effects of these compounds depends on the relative numbers of lesions in which inhibition of DNA synthesis can counteract their transforming effect.

Do men and women follow different trajectories to reach extreme longevity? Italian Multicenter Study on Centenarians (IMUSCE).

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in pro-inflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human life-span, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years.

Familial hypercholesterolemia study in Sardinia using 6 LDLR polymorphic markers based on PCR.

Twenty-two Sardinian families with multiple cases of hypercholesterolemia were investigated with six polymorphic markers of the low-density lipoprotein receptor (LDLR) gene that could be quickly analyzed by PCR-based methods. Five single nucleotide polymorphisms (SNP) in exons 8, 10, 13, 15, and 18 and a microsatellite marker flanking the 3' end of the LDLR gene were used to define the haplotypes at the LDLR locus for familial hypercholesterolemia (FH) diagnosis within families. No significant differences were observed between the allele frequencies of the normal and mutant chromosomes. In two families, hypercholesterolemia did not cosegregate with the LDLR locus. In the remaining 20 FH chromosomes, seven different haplotypes were identified. The same haplotypes were found with a similar frequency among the 61 normal chromosomes. Other five haplotypes were characteristic only of normal chromosomes. These data provide no evidence for a gene founder effect in the Sardinian population and, instead, highlight a pattern of genetic heterogeneity comparable with that found in mainland European populations. The replacement of the restriction fragment length polymorphisms currently used in the genetic analysis of FH with PCR-based markers proved to be a simple and less time-consuming method and did not reduce informativity in the molecular analysis of FH families.

Implication of Bcl-2 family genes in basal and D-amphetamine-induced apoptosis in preneoplastic and neoplastic rat liver lesions.

Molecular mechanisms of basal and D-amphetamine (AMPH)-induced apoptosis were studied in rat liver nodules, 12 (N12) and 30 (N30) weeks after initiation, and in hepatocellular carcinoma (HCC) induced by diethylnitrosamine in rats subjected to resistant hepatocyte model. Basal apoptosis in hematoxylin/eosin- and propidium iodide-stained sections was higher in nodules and HCC than in normal livers. It sharply increased in all tissues 4 hours after AMPH treatment (10 mg/kg), and declined to basal levels at 8 to 12 hours in liver and N12, but remained high up to 18 hours in N30 and HCC. c-myc, Tgf-alpha, p53, and Bcl-X(S) messenger RNA (mRNA) levels were higher, and Bcl-2 mRNA was lower in N12 and/or N30 and HCC than in normal liver. Four hours after AMPH injection, increase in c-myc and decreases in Bcl-2 and Bcl-X(L) mRNAs occurred in all tissues, whereas p53, Bax, and Bcl-X(S) mRNAs increased in N30 and HCC. These changes disappeared in liver and N12 at 18 hours, but persisted in N30 and HCC. c-Myc, P53, Bcl-2, and Bax proteins in normal liver and HCC +/- AMPH showed similar patterns. Tgf-beta1, Tgf-beta-RIII, CD95, and CD95L mRNA levels underwent slight or no changes in any tissue +/- AMPH. Basal Hsp27 expression was high in nodules and HCC, and was stimulated by AMPH in liver and N12, but not in N30 and HCC. These data suggest a role of dysregulation of Bcl-2 family genes and, at least in atypical lesions, of p53 overexpression, in basal and AMPH-induced apoptosis in nodules and HCCs. Hsp27 does not appear to sufficiently protect atypical lesions against apoptosis.

Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia : a study of patients with familial hypercholesterolemia from Sardinia.

One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the alpha-globin gene (alpha-thalassemia trait) and that 6% to 17% are beta-thalassemia carriers. In this population, a single mutation of beta-globin gene (Q39X, beta(0) 39) accounts for >95% of beta-thalassemia cases. Because previous studies have shown that Sardinian beta-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the beta-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76+/-1.08 mmol/L in subjects with beta(0)-thalassemia trait and 8.25+/-1.66 mmol/L in subjects without this trait (P<0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had beta(0)-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of beta(0)-thalassemia trait emerged also when we pooled the data from all FH subjects with and without beta(0)-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of beta(0)-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B-containing lipoproteins. The observation that our FH subjects with beta(0)-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of beta(0)-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.

Spectrophotometric measurement of hydroperoxides at increased sensitivity by oxidation of Fe2+ in the presence of xylenol orange.

The method, developed by modifying the FOX methods described by Wolff (Methods Enzymol. 233, 182-189, 1994), involves the oxidation of Fe2- by peroxides at low pH in the presence of both the ferric-complexing dye xylenol orange and sucrose, the amplifier of the reaction. The method proved to be a convenient, simple and efficient assay for the direct measurement of both water and lipid soluble peroxides. In fact it improves by about 60% the sensitivity of the FOX1 method for water soluble peroxides, and by 7-8 times that of the FOX2 method for lipid soluble peroxides. It allows the detection of 0.1 microM peroxide in the test solution. The method is suitable to measure the lipid hydroperoxides present in phosphatidylcholine liposomes and in human LDL. The data obtained allowed us to define a mathematical expression to calculate the lipid hydroperoxide content of liposomes knowing their oxidation index.

AKEntAnnos. The Sardinia Study of Extreme Longevity.

This paper describes an epidemiological study performed in all centenarians living in Sardinia, a large island located in the Mediterranean sea, 120 Km from the Italian coast. Due to its long-standing isolation, low immigration rate, high endogamy and rather uniform lifestyle, Sardinia offers an ideal setting in which to study the genetic traits associated with extreme longevity and successful aging. A total of 233 potentially eligible centenarians were traced in the entire territory. Of these, 66 died prior to being interviewed, 11 were not found and unknown, and 15 refused to be interviewed. A multidimensional home interview was administered to 141 centenarians, and an equivalent number of 60-year-old controls matched for gender and area of residence. Furthermore, 41 living siblings of the centenarians, and 41 age- and sex-matched controls for these siblings were also studied. The prevalence of centenarians was 13.56 per 100,000, and the female/male ratio was approximately 2. Prevalence and female/male ratio were consistent across the four Sardinian municipalities and are, respectively, higher and lower than those reported in other population-based surveys. A number of methodological problems confronted in doing the field work, and plans for future analysis of this rich dataset are discussed.

Correlation of c-myc overexpression and amplification with progression of preneoplastic liver lesions to malignancy in the poorly susceptible Wistar rat strain.

Persistent liver nodules (PNs) and hepatocellular carcinomas (HCCs) induced in F344 rats by the resistant hepatocyte (RH) model exhibit c-myc overexpression and amplification. The role of these changes in progression of PN was investigated in nodules with different propensities to evolve to HCC in resistant Wistar rats and, for comparison, in susceptible F344 rats. Initiation of rats with diethylnitrosamine was followed by selection with 2-acetylaminofluorene (AAF) plus partial hepatectomy (RH groups). Two additional Wistar rat groups received a second AAF treatment without (RH+AAF) and with a necrogenic dose of CCl4 (RH+AAF/CCl4) 15 d after selection. The number to liver ratio and volume of glutathione-s-transferase placental form-positive lesions were lower in the Wistar than the F344 RH groups 9 and 32 wk after initiation and increased after a second AAF cycle treatment with and without CCl4. DNA synthesis in glutathione-s-transferase placental form-positive lesions was low in Wistar RH group at 9 wk and was stimulated by additional AAF treatments. HCCs developed at 57-60 wk in F344 RH, Wistar RH+AAF, and RH+AAF/CCl4 rats. Tumor incidence and multiplicity were lower in RH+AAF rats than in RH+AAF/CCl4 and F344 rats. At 32 wk, PN exhibited c-myc overexpression that increased from RH to RH+AAF rats and to RH+AAF/CCl4 Wistar rats. This was associated with c-myc amplification in Wistar RH+AAF/CCl4 rats. These results showed correlation of c-myc overexpression and amplification with nodule propensity to progress to HCC in poorly susceptible Wistar rats and suggested a possible genetic mechanism for susceptibility to hepatocarcinogenesis. The experimental system used in this work may be a valuable tool for studies on molecular mechanisms underlying liver growth and tumorigenesis supported by c-myc overexpression.

Lack of influence of apolipoprotein E4 on lipoprotein levels in the island population of Sardinia.

BACKGROUND: In this study, the apolipoprotein E (apoE) genotypes as well as plasma lipid and lipoprotein levels have been determined for the first time in 633 healthy and unrelated inhabitants on the island of Sardinia (291 men and 342 women, age range 6-89 years), randomly selected in the four districts of Sardinia among healthy people having parents and grandparents born in the same geographic area. This island lies in the centre of the western Mediterranean sea with a well-recognized peculiar and preserved genetic background. The epsilon 3 allele frequency (0.897) is higher than that previously reported in southern and mainland Italy and is one of the highest in Europe and among Caucasians; epsilon 2 allele frequency (0.040) is the lowest in Europe; epsilon 4 (0.063) is unusually low and, most interestingly, does not change in the different age groups, i.e. does not decrease in old subjects. RESULTS: Lipid and lipoprotein analysis confirm that total cholesterol and low-density lipoprotein (LDL)-cholesterol levels are lower than those described in mainland Italy; moreover, in this large Sardinian series, accurately selected, epsilon 4 allele did not influence total cholesterol (P = 0.270), LDL-cholesterol (P = 0.667) and high-density lipoprotein (HDL)-cholesterol (P = 0.549) as in other populations. In contrast, subjects carrying the epsilon 2 allele were found to have significantly lower total cholesterol (P = 0.001) and LDL-cholesterol (P = 0.001) levels than epsilon 3 carriers. CONCLUSION: The low epsilon 4 frequency and the lack of influence of this allele on lipid metabolism is a unique characteristic and, to date, has been described only in Sardinia among Caucasians; it might contribute to the low prevalence of coronary artery disease in this island.