The proteasome as a druggable target with multiple therapeutic potentialities: Cutting and non-cutting edges.

Ubiquitin Proteasome System (UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs.

Alignment of diet prescription to genotype does not promote greater weight loss success in women with obesity participating in an exercise and weight loss program.

OBJECTIVE: Genetics contribute to variability in individual response to weight-loss interventions. The objective of this study was to determine the efficacy of a commercially available exercise and weight-loss program and whether alignment of diet to genotype related to lipid metabolism promotes greater success. DESIGN: Sedentary women with obesity (n = 63) had genotype (FABP2rs1799883, PPARG2rs1801282, ADRB3rs4994C3, ADRB2rs1042713, rs1042714) determined using a direct-to-consumer genetic screening kit purported to promote greater weight-loss success through dietary recommendations based on these genes. Participants were randomly assigned to follow a moderate carbohydrate (MC) or lower carbohydrate (LC) hypo-energetic diet that aligned (A) or did not align (NA) with genotype for 24 weeks while participating in a resistance training and walking program. Data were analysed by general linear model repeated measures adjusted for baseline variables and are presented as mean (95% confidence interval) changes from baseline. RESULTS: Participants in the LC group experienced greater improvements (p = 0.051, ηp 2 = 0.025) in per cent changes in body composition (weight: MC -3.32 [-1.4, -5.2], LC -5.82 [-4.1, -7.6]; fat mass: MC -7.25 [-3.2, -11.2], LC -10.93 [-7.3, -14.5]; fat-free mass: MC -0.32 [1.4, -2.0], LC -1.48 [0.7, -3.0]; and body fat percentage: MC -4.19 [-1.6, -6.8], LC -5.60 [-3.3, -7.9] %). No significant differences were observed between genotype groups (weight: A -5.00 [-3.3, -6.7], NA -4.14 [-2.2, -6.1]; fat mass: A -10.15 [-7.0, -13.6], NA -8.02 [-4.0, -12.0]; fat-free mass: A -1.23 [0.3, -2.8], NA -0.56 [1.12, -2.3]; and body fat: A -5.28 [-3.0, -7.6], NA -4.51 [-1.9, -7.1] %). CONCLUSIONS: Adherence to this exercise and weight-loss program promoted improvements in body composition and health outcomes. While individuals following the LC diet experienced greater benefits, alignment of these diets to this genetic profile did not promote greater health outcomes.

Protonation of aqueous alanine by photoionization of water.

Hydronium ions produced by photolysis of water are used to study the protonation dynamics of alanine zwitterions in water. The measurements are done by UV-VIS and UV-IR femtosecond transient absorption spectroscopy on alanine in H2O and D2O. It is estimated that the reaction rate constant for the deuteration of alanine zwitterions is 4 × 1010 M-1 s-1, while the reverse process has a rate constant of 2 × 108 s-1. In addition to hydronium ions the photolysis of water yields hydrogen atoms and hydrated electrons together with hydroxyl radicals and hydroxyl ions. However, no other products resulting from reactions between aqueous alanine and the photolysis products of water are positively identified during the first 530 ps after the photolysis. Potential secondary reactions that are not observed experimentally are discussed and an upper limit is set for their yield where possible.

Protein composition affects variation in coagulation properties of buffalo milk.

The aim of this study was to investigate the effects exerted by the content of casein and whey protein fractions on variation of pH, rennet-coagulation time (RCT), curd-firming time (K20), and curd firmness of Mediterranean buffalo individual milk. Measures of milk protein composition and assessment of genotypes at CSN1S1 and CSN3 were obtained by reversed-phase HPLC analysis of 621 individual milk samples. Increased content of αS1-casein (CN) was associated with delayed coagulation onset and increased K20, whereas average pH, RCT, and K20 decreased when ß-CN content increased. Milk with low κ-CN content exhibited low pH and RCT relative to milk with high content of κ-CN. Increased content of glycosylated κ-CN was associated with unfavorable effects on RCT. Effects of milk protein composition on curd firmness were less important than those on pH, RCT, and K20. Likely, this occurred as a consequence of the very short RCT of buffalo milk, which guaranteed a complete strengthening of the curd even in the restricted 31 min time of analysis of coagulation properties and for samples initially showing soft curds. Effects of CSN1S1-CSN3 genotypes on coagulation properties were not to be entirely ascribed to existing variation in milk protein composition associated with polymorphisms at CSN1S1 and CSN3 genes. Although the role of detailed milk protein composition in variation of cheese yield needs to be further investigated, findings of this study suggest that modification of the relative content of specific CN fractions can relevantly influence the behavior of buffalo milk during processing.

A new translocation t(1p;18) in an Italian Mediterranean river buffalo (Bubalus bubalis, 2n = 50) bull: cytogenetic, fertility and inheritance studies.

In recent years increasing attention has been paid to the cytogenetic control of Italian Mediterranean river buffalo (BBU) bulls authorized as sires which are registered in the stud book. Chromosome abnormalities described in this species are mainly numerical and affecting sex chromosomes. During routine cytogenetic analyses performed on young Italian Mediterranean river buffalo bulls in the progeny test, 1 animal was found to be carrier of a never before reported translocation t(1p;18) originated by fission of BBU1 and subsequent centric fusion of BBU1p with BBU18 as demonstrated by both R-banding and FISH-mapping techniques using specific molecular markers of BBU1p (DEFB1) and BBU18 (GPI). According to sperm analyses the semen characteristics were in physiological ranges, but the calf crop percentage was only 48.77% instead of 70-80%. Cytogenetic analyses performed on 50 offspring (36 females and 14 males) showed that 15 of them (30%) were carriers of the same translocation.

Short communication: CSN1S1-CSN3 (α(S1)-κ-casein) composite genotypes affect detailed milk protein composition of Mediterranean water buffalo.

The aim of the study was to investigate the effect of composite CSN1S1-CSN3 [α(S1)-κ-casein (CN)] genotype on milk protein composition in Mediterranean water buffalo. Content of α(S1)-CN, α(S2)-CN, ß-CN, γ-CN, κ-CN, glycosylated and unglycosylated κ-CN, α-lactalbumin, and ß-lactoglobulin was measured by reversed-phase HPLC using 621 individual milk samples. Genotypes at CSN1S1 and CSN3 were also obtained by reversed-phase HPLC. Two alleles were detected at CSN1S1 (corresponding to the A and B variants, O62823: p.Leu193Ser,) and at CSN3 (corresponding to the X1 and X2 variants, CAP12622.1: p.Ile156Thr). Increased proportions of α(S1)-CN in total casein (TCN) were associated with genotypes carrying CSN1S1 A. Genotypes associated with a marked decrease of the proportion of α(S1)-CN in TCN (composite genotypes AB-X1X1 and BB-X1X2) were associated with marked increases in the proportion of α(S2)-CN. In addition, composite genotypes carrying the X1 allele at CSN3 were associated with a greater proportion of α(S2)-CN in TCN relative to those carrying CSN3 X2. Composite genotypes greatly affected also the variability of ratios of κ-CN to TCN, with genotypes carrying the X1 allele at CSN3 being associated with decreased ratios. The decreased content of glycosylated κ-CN associated with CSN3 X1 was responsible for the overall lower content of total κ-CN in milk of X1-carrying animals. Increasing the frequency of specific genotypes might be an effective way to alter milk protein composition, namely the proportion of α(S1)-CN, α(S2)-CN, and κ-CN in TCN, and the degree of glycosylation of κ-CN.

Effect of parity, days in milk, and milk yield on detailed milk protein composition in Mediterranean water buffalo.

The effects of some nongenetic factors on milk protein fraction contents and relative proportions were estimated in 606 individual milk samples of Mediterranean water buffalo. Content of α(S1)-casein (CN), α(S2)-CN, ß-CN, γ-CN, κκ-CN, glycosylated κ-CN (glyco-κ-CN), α-lactalbumin, and ß-lactoglobulin was measured by reversed-phase HPLC. Relative contents of α(S1)-CN%, α(S2)-CN%, ß-CN%, and κ-CN% were, respectively, 32.1, 17.1, 34.5, and 15.7%, whereas γ-CN% accounted for 0.6% of total casein content. Increasing total casein content in milk would result in a greater proportion of ß-CN% at the expense of all of the other major casein fractions, especially of κ-CN%. Values of α(S2)-CN%, ß-CN%, and γ-CN% tended to decrease with parity, although their variations were not significant, whereas α(S1)-CN% and glyco-κ-CN% showed the opposite trend. Contents of most protein fractions showed the typical trends observed for milk components as lactation progressed, with high contents in early lactation, a minimum in midlactation, followed by a gradual increase toward the latter part of lactation. Values of α(S1)-CN% increased during lactation, whereas α(S2)-CN% decreased. The proportion of ß-CN% had its maximum value between 60 and 160 d of lactation, followed by a decrease, whereas κ-CN% had its minimum value in early lactation (<60 d) and remained relatively constant in the period of mid and late lactation. Glyco-κ-CN% and ß-lactoglobulin% decreased in the first part of lactation, to reach their minimum values in midlactation, followed by an increase. Milk of top-producing buffaloes, compared with that of low-producing ones, had a significantly greater value of ß-CN% and glyco-κ-CN%, and lower proportion of α(S1)-CN%. The possible effect exerted by protein genetic variants in affecting variation of milk protein fraction contents and relative proportions should be further considered to better get insight into buffalo milk protein composition.

Effect of CSN1S1-CSN3 (α(S1)-κ-casein) composite genotype on milk production traits and milk coagulation properties in Mediterranean water buffalo.

The aim of this study was to estimate effects of CSN1S1-CSN3 (α(S1)-κ-casein) composite genotypes on milk production traits and milk coagulation properties (MCP) in Mediterranean water buffalo. Genotypes at CSN1S1 and CSN3 and coagulation properties [rennet clotting time (RCT), curd firming time (K20), and curd firmness (A30)] were assessed by reversed-phase HPLC and computerized renneting meter analysis, respectively, using single test-day milk samples of 536 animals. Alternative protein variants of α(S1)-CN and κ-CN were detected by HPLC, and identification of the corresponding genetic variants was carried out by DNA analysis. Two genetic variants were detected at CSN1S1 (A and B variants) and 2 at CSN3 (X1 and X2 variants). Statistical inference was based on a linear model including the CSN1S1-CSN3 composite genotype effect (7 genotypes), the effects of herd-test-day (8 levels), and a combined days in milk (DIM)-parity class. Composite genotype AB-X2X2 was associated with decreased test-day milk yield [-0.21 standard deviation (SD) units of the trait] relative to genotype BB-X2X2. Genotypes did not affect milk protein content, but genotype AB-X1X1 was associated with increased fat content compared with genotype BB-X2X2 (+0.28 SD units of the trait) and AB-X1X1 (+0.43 SD units of the trait). For RCT, the largest difference (+1.91 min; i.e., 0.61 SD units of the trait) was observed between genotype AA-X1X2 and AB-X1X1. Direction of genotype effects on K(20) was consistent with that for RCT. The maximum variation in K20 due to genotype effects (between AA-X1X2 and AB-X1X1 genotypes) was almost 0.9 SD units of the trait. Magnitude of genotype effects was smaller for A30 than for RCT and K20, with a maximum difference of 0.5 SD units of the trait between genotype AA-X1X2 and AA-X1X1. The B allele at CSN1S1 was associated with increased RCT and K20 and with weaker curds compared with allele A. Allele X2 at CSN3 exerted opposite effects on MCP relative to CSN1S1 B. Because of linkage disequilibrium, allele B at CSN1S1 and allele X2 at CSN3 tend to be associated and this likely makes their effects cancel each other. This study indicates a role for casein genes in variation of MCP of buffalo milk. Further studies are necessary to estimate the effects of casein genetic variants on variation of cheese yield.

Chromosome fragility in river buffalo cows exposed to dioxins.

Fifty river buffalo (Bubalus bubalis, 2n = 50) cows reared in two different provinces of Campania (southern Italy) underwent cytogenetic investigations to ascertain possible differences in their chromosome stability. One group (Caserta province) was under legal sequestration due to the presence in the milk mass of higher mean values of dioxins [21.79 pg/g of fat as sum of polychloro-dibenzo-dioxins (PCDDs), polychloro-dibenzo-furans (PCDFs) and dioxin-like polychlorobiphenyls (DL-PCBs)] than both those permitted (6.0 pg/g of fat as WHO-TEQ) and those (1.3 pg/g of fat as WHO-TEQ) observed in the control group raised in Salerno province. Two types of peripheral blood cell cultures were performed: without (normal cultures for the chromosome abnormality (CA) test: chromatid breaks, chromosome breaks, fragments) and with the addition of BrdU for the sister chromatid exchange (SCE) test). The CA test revealed a significantly (P < 0.01) higher chromosome fragility in the exposed cows compared to the control. Indeed, mean values of CA/cell were 1.26 ± 1.15 in exposed cows and 0.37 ± 0.71 in the control. Mean SCE was higher in exposed cows (8.50 ± 3.35) than that (8.29 ± 3.51) found in the control but the difference was not significant. Comparison within the same group of cows at first (FL) and multiple (ML) lactations revealed significantly (P < 0.01) higher mean values of CA/cell in exposed ML-cows vs FL-cows while no statistical differences were found between ML-cows and FL-cows in the control farm. By contrast, significantly (P < 0.01) higher mean values of SCE were found in both groups of FL-cows versus ML-cows. Comparisons with other previous studied species (sheep and cattle) were also performed.

Short communication: Translational efficiency of casein transcripts in Mediterranean river buffalo.

Buffalo milk is characterized by the presence of all 4 casein fractions (α(S1), ß, α(S2), and κ) encoded by the 4 tightly linked autosomal genes (CSN1S1, CSN2, CSN1S2, and CSN3, respectively). In the present paper, we report for the first time a quantitative characterization of buffalo casein transcripts and show that the 4 genes are not transcribed and translated with the same efficiency. In particular, the analysis of individual milk samples obtained from 9 Mediterranean river buffaloes showed that the most abundant casein fractions were ß (53.45%) and α(S1) (20.61%), followed by α(S2) and κ, at 14.28 and 11.66%, respectively. Quantification of the corresponding mRNA showed that the percentage of transcripts of the 4 caseins was 16.48, 23.18, 55.87, and 4.47% for α(S1), ß, α(S2), and κ, respectively. Translation efficiency was 0.25 for CSN1S2, 1.31 for CSN1S1, 2.39 for CSN2, and 2.69 for the CSN3 transcripts, respectively. A comparison of nucleotide sequences with the Kozak consensus sequence was also carried out to investigate if the mRNA sequences might be responsible for the observed differences.

Sequence analysis and genetic variability of stearoyl CoA desaturase (SCD) gene in the Italian Mediterranean river buffalo.

Stearoyl-CoA desaturase (SCD) plays a key metabolic role by changing the saturated FA content of ruminant milk and meat. In this study we characterized for the first time the stearoyl-CoA desaturase (SCD) gene in river buffalo (Bubalus bubalis) and investigated its genetic variability. On a total of 78 buffaloes, 15 SNPs were detected and 6 of them were preliminarily genotyped. In particular, the g.133A>C SNP was found to create a new consensus site for the SP1 binding site, thus generating a new tandem repeat in the promoter region. A preliminary association study with the milk fatty acid content showed that the C allele significantly affects the total desaturation index (P<0.01). Linkage disequilibrium analysis allowed identification of 7 haplotypes and 4 tag SNPs. Such polymorphisms could represent useful genetic markers for association studies with fatty acid composition, but further studies are needed to evaluate their potential use to improve the nutritional quality of the dairy products.

Clinical trials update from the American Heart Association meeting: PROSPER, DIAL, home care monitoring trials, immune modulation therapy, COMPANION and anaemia in heart failure.

This article continues a series of reports on research developments of particular interest to those involved in the management of patients with heart failure. Summaries of the following trials, reported at the 75th Scientific Sessions of the American Heart Association held in Chicago, Illinois between 17th and 20th November 2002 are included: PROSPER; DIAL; home care monitoring trials; immune modulation therapy; COMPANION; and anaemia in heart failure.

Clinical trials update from the European Society of Cardiology: CARMEN, EARTH, OPTIMAAL, ACE, TEN-HMS, MAGIC, SOLVD-X and PATH-CHF II.

This article continues a series of reports on research developments related to the field of heart failure. Reports of presentations made at the Hot Line sessions of the European Society of Cardiology XXIV Congress held in Berlin, Germany, between 31 August and 4 September 2002 are included. Summaries of the results of the following trials are presented: CARMEN, EARTH, OPTIMAAL, ACE, TEN-HMS, MAGIC, SOLVD-X and PATH-CHF II.

Clinical trials update: RENEWAL (RENAISSANCE and RECOVER) and ATTACH.

This article continues a series of reports on recent research developments in the field of heart failure. Reports of two key presentations made at the European Society of Cardiology Heart Failure Update meeting held in Oslo, Norway from 8 to 11 June 2002, are included in this article. Summaries of the results of the RENEWAL (RENAISSANCE and RECOVER) and ATTACH studies, presented at the Hot Line sessions held during the meeting are reported.

Clinical trials update: highlights of the scientific sessions of the XXIII Congress of the European Society of Cardiology--WARIS II, ESCAMI, PAFAC, RITZ-1 and TIME.

This article continues a series of reports updating recent research developments of particular interest to personnel involved in the treatment and management of patients with heart failure. This is a summary of selected presentations made at the Scientific Sessions of the XXIII Annual Congress of the European Society of Cardiology. Summaries of the following clinical studies are included: WARIS-II, ESCAMI, PAFAC, RITZ-I and TIME.

Clinical trials update: Highlights of the Scientific Sessions of Heart Failure 2001, a meeting of the Working Group on Heart Failure of the European Society of Cardiology. CONTAK-CD, CHRISTMAS, OPTIME-CHF.

This article continues a series of reports summarising recent research developments pertinent to the topic of heart failure. This is a summary of presentations made at Scientific Sessions of Heart Failure 2001, a meeting of the Working Group on Heart Failure of the European Society of Cardiology. Clinical studies of particular interest to people caring for patients with heart failure include CONTAK-CD, CHRISTMAS and further updates on OPTIME-CHF. A brief review of the current status of cardiac resynchronisation therapy is included.

DNA aberrations in urinary bladder cancer detected by flow cytometry and FISH: prognostic implications.

We evaluated the genetic changes in bladder cancer biopsy by fluorescence in situ hybridization (FISH) and related them to stage and grade of the tumor, ploidy (FCM) and clinical outcome, to determine a simple method to identify tumors with a poorer prognosis. Using FISH the numerical aberrations of chromosomes 1, 7, 9, 17 in tumor's imprints of 70 patients with transitional cell cancer (TCC) were determined. First of all, the data demonstrated that the sensitivity of FISH in detecting quantitative DNA aberrations exceeds FCM's sensitivity. The frequency of chromosome 1 and 9 aberrations did not show significant differences in diploid and aneuploid tumors in different stage and grade. On the contrary, the chromosome 7 and 17 aneusomy showed greater differences between pT1 and pT2-3 tumors (p<0.032 and p<0.0006, respectively) than between stage pTa and pT1. In our investigation, an increasing number of aberrations was observed in all chromosomes examined in tumors of patients who afterwards underwent cystectomy and/or had recurrent tumors. These results suggest that chromosome 7 and 17 aneusomy could be predictive of adverse outcome in a subgroup of patients with superficial tumors at presentation.

Investigation of genetic alterations associated with development and adverse outcome in patients with astrocytic tumor.

Models describing progression in the genetic derangement of glial tumors have shown chromosomal loss and gain occurring most frequently in high-grade lesions, suggesting that identification of these aberrations may be prognostically significant. In this study, Fluorescence in situ hybridization (FISH) has been used to determine, and to confirm, loss and gain of chromosomes 1, 8, 10, 12 and 17, in formalin-fixed, paraffin-embedded brain biopsy tissue taken from 60 brain gliomas submitted to surgical resection or stereotactic biopsy. FISH analysis may be a valuable adjunct to histological grading. The results showed that this molecular cytogenetic technique is an important clinical and experimental tool that provides new insight on genetic alterations, confirming gain and loss of genetic material that occurs at the initiation and progression of human glioma. Our data suggests that potentially useful prognostic information may be obtained through this approach. Monosomy 10 was the most statistically significant negative predictor of patient survival, showing a significant correlation with the histological grading.