Short communication: In vitro rumen gas production and starch degradation of starch-based feeds depend on mean particle size.

Our objective was to model the effect of mean particle size (mPS) on in vitro rumen starch degradation (IVSD) and the kinetics of gas production for different starch-based feeds. For each feed, 2 batches of the same grains were separately processed through 2 different mills (cutter or rotor speed mills), with or without different screens to achieve a wide range of mPS (0.32 to 3.31 mm for corn meals; 0.19 to 2.81 mm for barley meals; 0.16 to 2.13 mm for wheat meals; 0.28 to 2.32 mm for oat meals; 0.21 to 2.36 mm for rye meals; 0.40 to 1.79 for sorghum meals; 0.26 to 4.71 mm for pea meals; and 0.25 to 4.53 mm for faba meals). The IVSD data and gas production kinetics, obtained by fitting to a single-pool exponential model, were analyzed using a completely randomized design, in which the main tested effect was mPS (n = 6 for all tested meals, except n = 7 for corn meals and n = 5 for sorghum meals). Rumen inocula were collected from 2 fistulated Holstein dairy cows that were fed a total mixed ration consisting of 16.2% crude protein, 28.5% starch, and 35.0% neutral detergent fiber on a dry matter basis. The IVSD, evaluated after 7 h of rumen incubation, decreased linearly with increasing mPS for corn, barley, wheat, rye, pea, and faba meals, and decreased quadratically with increasing mPS for the other meals. The y-axis intercept for 7-h IVSD was below 90% starch for corn, barley, and rye feeds and greater than 90% for the other tested feeds. The mPS adjustment factors for the rate of rumen starch degradation varied widely among the different tested feeds. We found a linear decrease in starch degradation with increasing mPS for barley, wheat, rye, and pea meals, whereas we noted a quadratic decrease in starch degradation for the other tested meals. Further, we observed a linear decrease in the rate of gas production with increasing mPS in each tested feed, except for pea meal, which had a quadratic relationship. For each 1 mm increase in mPS, the gas production was adjusted by -0.009 h-1 for corn, -0.011 h-1 for barley, -0.008 h-1 for wheat, and -0.006 h-1 for faba, whereas numerically greater adjustments were needed for oat (-0.022 h-1), rye (-0.017 h-1), and sorghum (-0.014 h-1). These mPS adjustment factors could be used to modify the starch-based feed energy values as a function of mean particle size, although in vivo validation is required.

Technical note: Relationship between in situ NDF degradability and enzymatic NDF hydrolysis in forages, nonforage fibrous feeds, and crop residues.

The study was performed on forages ( = 8), nonforage fibrous feeds ( = 10), and crop residues ( = 2). Samples were characterized for in situ NDF degradability (NDFD) at 6, 12, 18, 24, 30, 36, 48, 72, 96, 120, and 240 h of ruminal incubation. Then, samples were characterized for enzymatic NDFD by adopting a multistep enzymatic method consisting of a preincubation (PreInc) phase followed by enzymatic incubation (EnzInc) steps. In the PreInc phase, samples were incubated in a NaOH solution for 0, 30, 60, or 90 min. Then, in the EnzInc phase, samples were first incubated in a buffered enzymatic solution containing hemicellulase, cellulase, and Viscozyme L enzymes. Then, samples were incubated in a xylanase-buffered enzymatic solution. These 2-step EnzInc lasted for a total of 16 (8 h for the first enzymatic step + 8 h for the second enzymatic step), 32 (16 + 16 h), or 48 h (24 + 24 h). The enzymatic NDFD coefficients were increased by increasing both PreInc and EnzInc incubation times, and no PreInc × EnzInc interaction was observed, except for ryegrass hay. On average, enzymatic NDFD increased ( < 0.05) by 0.35, 0.54, or 0.68, respectively, for 30-, 60-, or 90-min PreInc compared with 0-min PreInc. The enzymatic NDFD increased ( < 0.05), on average, by 0.11 in 32-h EnzInc or 0.16 in 48-h EnzInc with respect to 16-h EnzInc. Enzymatic NDFD were used to predict in situ NDFD coefficients by adopting single regression equations. High coefficients of regression ( > 0.80, < 0.05) and low errors of prediction were measured when specific enzymatic conditions were performed to predict in situ NDFD at intermediate (from 24 to 48 h) ruminal incubation. Generally, worse regression performances were obtained when enzymatic NDFD were used to predict in situ NDFD evaluated after shorter or longer incubation times. The direct prediction of the rate of NDF degradation was not possible using enzymatic NDFD coefficients. Even if the proposed multistep enzymatic method appeared promising, further studies are required to improve enzymatic NDFD prediction ability within specific forage types or nonforage fibrous feeds.

Short communication: The effect of an exogenous enzyme with amylolytic activity on gas production and in vitro rumen starch degradability of small and large particles of corn or barley meals.

The objective of this study was to evaluate the effect of exogenous amylase supplementation on gas production and on in vitro rumen starch degradability (IVSD) of different sized particles of corn and barley meals (Cm and Bm, respectively). An aqueous liquid amylase formulation from Bacillus licheniformis was tested at 3 enzyme doses (EnzD; 0, 300 and 1,500 kilo novo units/kg of dry matter) on small (<750 µm) and large (≥750 µm) particle size (PS) of Cm and Bm. Data were analyzed according to a randomized complete block design with a factorial arrangement of treatments; the main tested effects were PS, EnzD, and their interaction. Fermentation run entered in the model as random effect. The mixed rumen fluid was collected from 2 rumen-fistulated Holstein dry dairy cows fed at maintenance (forage:concentrate ratio of 80:20; 12% crude protein; 55% amylase-treated neutral detergent fiber). Small particles of both Cm and Bm had a greater rate of fermentation and shorter lag time than large particles. The rate of starch degradation was greater for small than for large particles of Bm, being 0.187 and 0.125 1/h, respectively. Conversely, the rate of starch degradation of Cm averaged 0.063 1/h and was similar among treatments. Enzyme supplementation tended to reduce lag time and to increase rate of fermentation for both PS of Cm and Bm, with a more pronounced effect for small PS. A limited EnzD effect was measured for IVSD data and rate of starch degradation; PS influenced fermentation parameters and the magnitude of starch degradation more than EnzD. Supplementation with exogenous amylase influenced the rumen fermentation pattern of small and large PS of Cm and Bm, even if the effect of the enzyme supplementation differed according to the PS of cereal meals.

Gas production and starch degradability of corn and barley meals differing in mean particle size.

The objective of this study was to verify the effect of mean particle size (mPS) on both gas production and in vitro rumen starch degradability (IVSD) of corn and barley meals (Cm and Bm, respectively). Batches of the same Cm or Bm were separately processed through 2 different mills (i.e., a cutter mill or a rotor speed mill) equipped with or without different screens to achieve different mPS for each tested meal. Samples were analyzed accordingly to a completely randomized design and the main tested effect of model was mPS (n=11, from 0.46 to 3.50mm mPS for Cm or n=10, from 0.11 to 2.98mm mPS for Bm). For both in vitro assays, the rumen inocula were collected from 2 rumen-fistulated Holstein lactating dairy cows fed a total mixed ration with 16.2% crude protein, 28.5% starch, and 35.0% neutral detergent fiber on a dry matter basis. To fit gas production data, 1-pool exponential model and 1-pool or 2-pool Gompertz models were adopted. The rate of gas production decreased and lag increased by increasing mPS of both Cm and Bm, irrespective of adopted 1-pool models. When the 2-pool Gompertz model was used to fit gas production data, a shift of particles from fast to slow fermentable pools was measured by increasing mPS. In particular, the ratio between fast and slow final volumes ranged from 0.90 at 0.11mm mPS to 0.10 at 2.98mm mPS for Bm. For Cm, the ratio between fast and slow final volumes decreased quadratically by increasing mPS, with the highest value (i.e., 0.58) measured at the lowest tested mPS. Values lower than 0.10 were measured for mPS greater than 1.93mm for Cm. Concerning IVSD data, linear decreases in rate of starch degradation equal to -0.049 or -0.092h(-1) for each 1-mm increase in mPS were achieved for Cm and Bm, respectively. The 7-h IVSD decreased by 6.3 or 6.5% starch for each 1-mm increase in mPS of Cm or Bm, respectively. Present findings supported the hypothesis that different particle sizes within the same starch source represent an important factor influencing both fermentation kinetic parameters and IVSD.

Effect of the inclusion of dry pasta by-products at different levels in the diet of typical Italian finishing heavy pigs: Performance, carcass characteristics, and ham quality.

The effect of pasta inclusion in finishing pig diets was evaluated on growth performance, carcass characteristics, and ham quality. Pigs (144) were assigned to 4 diets with different pasta levels: 0 (control, corn-based diet), 30, 60, or 80%. Pigs fed pasta had greater (linear, P<0.01) feed intakes than controls. Pasta increased (quadratic, P<0.01) carcass weight and dressing percentage reaching the highest values at 30% inclusion level, and reduced (linear, P<0.01) the Longissimus thoracis et lumborum thickness. Pasta decreased (linear, P<0.01) linoleic acid and polyunsaturated fatty acid levels in subcutaneous (fresh and seasoned hams) and intramuscular (seasoned hams) fat, and enhanced saturated fatty acid content in subcutaneous fat (fresh hams: quadratic, P<0.01; seasoned hams: linear, P=0.03). Proteolysis index, colour, weight losses, and sensory properties (excepted extraneous taste) of the hams were unaffected by the pasta. Pasta could be considered as an ingredient in the diet for typical Italian finishing heavy pigs.

Effect of replacing corn with hulled and hulless or low-amylose hulless barley varieties on growth performance and carcass quality of Italian growing-finishing pig.

A study was conducted to evaluate the effect of diets based on hulled or hulless (normal- and low-amylose) barley varieties on growth performance and carcass characteristics in heavy growing-finishing pigs for the production of protected designation of origin (PDO) Italian products. The study was performed with 40 gilts and 40 barrows (Italian Duroc × Italian Large White). Four diets were formulated: 1) corn-based diet (control), 2) control diet with 80% of a normal-amylose hulled barley variety named Cometa (Cometa), 3) control diet with 80% of a normal-amylose hulless barley variety named Astartis (Astartis), and 4) control diet with 80% of a low-amylose hulless barley variety named Alamo (Alamo). The diets were formulated according to 3 growth phases (P1, 40 to 80 kg BW; P2, 80 to 120 kg BW; and P3, 120 to 170 kg BW), with the same Lys:DE ratio (2.60, 2.20, and 1.80, respectively in P1, P2, and P3) according to the NRC requirements for P1 and P2 and according to requirements for high-performing pigs for P3. The diets were analyzed for their in vitro starch digestion potentials (predicted glycemic index, pGI) and for their resistant starch (RS) contents. In P1, P2, and P3, the Alamo diet had the numerically lowest RS contents and greatest pGI values, whereas the control diet had the numerically greatest RS contents and the lowest pGI values. Throughout the study, the pigs fed Cometa and Alamo diets grew faster (P < 0.01) than those fed the control diet, whereas pigs receiving Astartis diet grew in a similar manner to those receiving all the other diets. Pigs fed Cometa and Alamo achieved greater final BW (P < 0.01) compared with those fed the control diet. The pigs receiving the Astartis diet had a mean final BW similar to that of the pigs fed other diets. Throughout the study, the control group had a lower grams per megacalorie DE (P < 0.01) compared with the pigs fed diets with barley, whereas the gain per megacalorie of DE (G/Mcal DE) was greater (P < 0.01) for the pigs fed hulled barley compared with the pig fed hulless barleys. No difference in carcass characteristics was found among treatments (P > 0.05). This study showed that diets based both on hulled and hulless barley might be suitable for the heavy pig breeding intended to the production of Italian PDO products. In addition, hulled or low-amylose hulless barley could be valuable to support maximum pig growth performance without affecting carcass composition.

Addition of nonstarch polysaccharides degrading enzymes to two hulless barley varieties fed in diets for weaned pigs.

A study was conducted to evaluate the effect of 2 hulless barley varieties, with or without the addition of a nonstarch polysaccharide (NSP) enzyme complex (ß-glucanase and xylanase), on growth performance of weaned piglets in a 42-d feeding study. The study was conducted with 140 piglets (PIC × Duroc). Pigs were allocated to pens (4 castrated males or 4 females per pen) based on BW and sex, and pens were assigned to 5 experimental diets with 4 pens of castrated males and 3 pens of females per treatment. Five isonitrogenous and isoenergetic diets were compared: 1) control corn-based diet (CTR), 2) diet with corn and wheat bran replaced by the Astartis hulless barley variety (AS), 3) diet with corn and wheat bran replaced by the AS supplemented with the NSP enzyme complex (ASE), 4) diet with corn and wheat bran replaced by the Alamo hulless barley variety (AL), and 5) diet with corn and wheat bran replaced by the AL supplemented with the NSP enzyme complex (ALE). The diets were formulated to meet or exceed nutrient requirements and offered in 2 phases: d 0 to 14 and d 14 to 42. At the end of the study, pigs fed AS and AL had equal weights as pigs fed CTR. Pigs fed the hulless barley diets had greater (P < 0.05) ADG during the second phase (P2) and overall phase, BW at d 42, and G:F during the P2 than those fed the CTR. Pigs fed the ASE and ALE had greater (P < 0.05) ADFI during the P2 and overall ADG than those fed the AS and AL. The increases in ADG during the P2 and final BW obtained with NSP enzyme supplementation were greater in pigs fed the AS than those fed the AL (barley × enzyme, P < 0.05). On the other hand, the NSP enzyme complex increased G:F in pigs fed the AS during the P2 and overall phase, but it had no effect on those fed the AL (barley × enzyme, P < 0.05). In conclusion, hulless barley with or without the NSP enzyme complex can be a replacement ingredient for corn and wheat bran in weaned pig diets. Addition of the NSP enzyme complex to AS variety, but not AL variety, improved growth performance of weanling pigs.

In vitro production of short-chain fatty acids from resistant starch by pig faecal inoculum.

The need to improve the knowledge of fermentation processes within the digestive tract in pigs is growing, particularly for ingredients that may act as potential prebiotic sources, such as resistant starch (RS). A study (based on enzymatic digestion followed by in vitro fermentation) was conducted to investigate whether various sources of RS, obtained from eight native starches characterized by inherent heterogeneous starch chemistry and structure, can influence short-chain fatty acid (SCFA) concentrations and relative production kinetics. Total and individual SCFA productions were evaluated over time and up to 72 h of incubation. The in vitro hydrolysis of native starches allowed a classification from very high [≥ 650 g/kg dry matter (DM)] to low (<50 g/kg DM) RS amount. The total SCFA production was similar between ingredients, whereas acetate and butyrate molar ratios in the SCFA profile differed (from 0.48 to 0.56 and from 0.17 to 0.25, respectively; P < 0.05). Differences in fermentation kinetic parameters for total and individual SCFA productions were observed (P < 0.05). Considering the total SCFA production after 72 h of incubation, the time at which half of the maximum production has been reached (T 1/2), the maximum rate of production (R max) and its time of occurrence (T max) differed between ingredients (P < 0.05), with values ranging from 6.1 to 11.9 h, from 0.459 to 1.300 mmol/g DM incubated per hour and from 5.1 to 9.8 h, respectively. Overall, a similar trend was observed considering individual SCFA productions. In particular, T 1/2 ranged from 6.4 to 12.5 h, from 5.5 to 12.5 h and from 6.7 to 11.3 h for acetate, propionate and butyrate, respectively (P < 0.05). For R max, differences were obtained for propionate and butyrate productions (P < 0.05), whereas no difference was recorded for acetate. In summary, our findings indicated that both quantitative and qualitative production of SCFA and related kinetics were influenced by fermentation of RS obtained from native starches characterized by heterogeneous starch characteristics. Current findings are based on an in vitro approach, and thus require further in vivo validations.

Plasma glucose response and glycemic indices in pigs fed diets differing in in vitro hydrolysis indices.

Different dietary starch sources can have a great impact in determining starch digestion potential, thus influencing the postprandial blood glucose response. Our objectives were to define: (i) the incremental plasma glucose response in pigs fed diets containing various sources of starch differing in in vitro digestion patterns, (ii) the in vivo glycemic index (GI) values for the same diets, (iii) the possible relationship between in vitro and in vivo data. Diets, formulated with 70% of starch from five heterogeneous sources, were characterized in depth by using two distinct in vitro evaluations. The first one was based on the Englyst-assay for nutritional classification of starch fractions, whereas the second one was based on a time-course multi-enzymatic assay up to 180 min from which the hydrolysis indices (HIs) were calculated and used as a link between the physicochemical properties of starch from diets and the in vivo responses. For the in vivo study, five jugular-catheterized pigs (35.3 ± 1.1 kg body weight) were fed one of the five diets for 6-day periods in a 5 × 5 Latin square design. On day 5, blood was collected for 8 h postprandially for evaluating glucose appearance. On day 6, blood was collected for 3 h postprandially for the estimation of the GI. Starchy diets differed for rapidly digestible starch (from 8.6% to 79.8% of total starch (TS)) and resistant starch contents (from 72.5% to 4.5% of TS). Wide between-diets variations were recorded for all the kinetic parameters and for the HI calculated from the in vitro digestion curves (P < 0.05). On the basis of the obtained HI, diets contained starch with a very low to a very high in vitro digestion potential (ranging from 26.7% to 100.0%; P < 0.05). The glucose response differed among diets (P < 0.05), with marked differences between 15 and 120 min postprandial. Overall, the ranking of incremental glucose appearance among diets agreed with their in vitro HI classification: high HI diets increased plasma glucose response more (P < 0.05) than low HI diets. Lastly, different in vivo GIs were measured (ranging from 30.9% to 100.0%; P < 0.05). The relationship between HI and GI showed a high coefficient of determination (R2 = 0.95; root mean square error (RMSE) = 15.8; P < 0.05). In conclusion, diets formulated with starches with a wide range in HI potential can strongly affect the postprandial glucose response in pigs.

Technical note: quantification of zeins from corn, high-moisture corn, and corn silage using a turbidimetric method: comparative efficiencies of isopropyl and tert-butyl alcohols.

Zeins are corn endosperm storage proteins that encapsulate starch granules into a protein matrix, which can act as a barrier to starch accessibility and digestion. Laboratory methods to quantify zein are seldom used because they are considered arduous and time-consuming. A recently published rapid turbidimetric method (mTM) was reinvestigated by changing the solution originally used for the zein solubilization step. In particular, the aim was to explore whether, and to what extent, the use of tert-butyl alcohol (t-BuOH-mTM) in lieu of isopropyl alcohol (i-PrOH-mTM) was able to improve the quantification of zeins from dry corn, high-moisture corn, and corn silage samples. The nature of the alcohol influenced the zein extraction values, and t-BuOH-mTM gave higher zein values in corn (3.6 vs. 3.3 g/100 g of dry matter) and corn silage samples (1.2 vs. 0.9 g/100 g of dry matter) compared with i-PrOH-mTM. In contrast, similar zein extraction values were obtained for high-moisture corn (2.1 vs. 1.9 g/100 g of dry matter, respectively). Sodium dodecyl sulfate-PAGE analysis revealed no contamination by nonzein proteins with the use of tert-butyl alcohol. Overall, these findings indicated that tert-butyl alcohol has a greater ability to solubilize zein compared with isopropyl alcohol and thus the t-BuOH-mTM allowed greater extraction of zeins. Considering the growing interest of animal nutritionists in zein proteins, such results should provide useful information for routine laboratory analysis.

Oxidative stress and ERK1/2 phosphorylation as predictors of outcome in hepatocellular carcinoma patients treated with sorafenib plus octreotide LAR.

We reported a relevant activity of the combination between sorafenib and octreotide long-acting release (LAR) in advanced hepatocellular carcinoma (HCC) patients. In this work, we have studied if oxidative stress in both serum and peripheral blood mononuclear cells (PBMC) and pERK activation status in PBMC could be predictive of response. In the 20 responsive patients, the decrease of reactive oxygen species levels was already detectable after 10 days (T10) from the beginning of sorafenib administration, and this effect was enhanced by the combined treatment with sorafenib+octreotide LAR (T21). This effect correlated with the modulation of superoxide dismutase (SOD) activity (physiological scavenger of O(2-)) and of serum nitric oxide (NO) levels. Sorafenib alone induced an increase of about 40% of NO levels and of about two-fold of SOD activity in responsive patients, and both effects were significantly potentiated by the combined treatment. We found a gradual reduction of Erk1/2 activity, as evaluated by cytofluorimetric analysis, in 15 responsive patients reaching about 50% maximal decrease at T21. On the other hand, in 17 resistant patients, Erk1/2 activity was about 80% increased at T21. The determination of both the oxidative stress status and pERK activity in PBMC has high value in the prediction of response to sorafenib+octreotide therapy in HCC patients.

Possible antioxidant role of SPA therapy with chlorine-sulphur-bicarbonate mineral water.

The aim of our research was to analyze the antioxidant role and efficacy of thermal or salus per aquam (spa) therapy with chlorine-sulphur-bicarbonate mineral water. The study has been performed on 30 rats. The animals were randomized in three groups, each of them composed by ten animals, denominated A, B and C. The A group was the control group and was not subjected to any specific treatment (placebo); the B group has been treated with a standard cycle of hydropinics treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated STABIA; the C group was treated with a standard cycle of hydropinic treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated SULFUREA. After two weeks of treatment all the rats were sacrificed and blood was collected for the plasmatic determination of reactive oxygen species (ROS). The results demonstrated a significant (P < 0.05) reduction of ROS in B (374 Carr. U. +/-73) and C group (399 carr. U. +/-62) treated with mineral waters if compared with control group (571 + 69 Carr. U.). In conclusion this study suggests a possible antioxidant effect of chlorine-sulphur-bicarbonate spa hydropinic treatment with a consequent suitable intestinal physiology, with reduction of the functional and organic modifications that can lead to pathological disorders of the gastroenteric diseases in whose pathogenesis the oxidative stress can develop an important role.

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-alpha effects in inducing apoptosis on human cancer cells through the generation of oxidative stress.

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution.

Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products.

A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.

Alternative therapy of earth elements increases the chondroprotective effects of chondroitin sulfate in mice.

The administration of mineral sulphur water is an alternative experimental approach for the treatment of rheumatic diseases, such as osteoarthritis (OA), that cause the degeneration of bone and cartilage and sufferance to the patients. Chondroitin sulfate (CS) is a symptomatic slow acting nutropeucital agent currently used in molecular therapy of OA. Therefore, we have studied the role and efficacy of the selective soil paste from the mineral sulphur enriched spring (mud)-therapy alone or in combination with CS in the treatment of OA. The study was performed on 40 C57 Black 6N mice, an experimental model which spontaneously develop an osteoarthritic process. The animals were divided in 4 groups and were treated with the single agents or with the combination. After 30 days of treatment all the mice were sacrificed and right knees and blood were collected. It was found that CS determined a reduction of radiological and histological features of chondrodegeneration and that mud-therapy increased the effects of CS in the animal group treated with the combination. However, the effects of thermal therapy alone were not statistically significant. Since OA is characterized by an increase of the production of nitric oxide (NO) by chondrocytes in extracellular matrix with its consequent elevation in serum and synovial fluid, we have evaluated the effects of the treatments on serum NO levels. CS alone induced a statistically significant reduction of NO serum levels (90+/-13 micromM vs 219+/-60 microM of control group, P<0.05) while mud-therapy alone induced a not statistically significant reduction of serum NO (170+/-62 microM, P>0.05). However, the latter strongly potentiated the decrease of serum NO induced by CS (31+/-1.5 microM) with a high statistical significance if compared to both the control group (P<0.01) and the CS-treated group (P<0.05). In conclusion, this study demonstrates that mud-therapy with sulphur mineral water could represent an important phase of the therapeutic strategy of OA. This experimental strategy could integrate and potentiate the standard pharmacological tools. Moreover, we have set a valid experimental in vivo model for the study of the thermal effects on the development of OA.

Docetaxel induces p53-dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells.

Docetaxel (Taxotere, DTX) is a promoter of apoptosis in cancer cells. Since cytotoxic mechanisms of DTX are not yet fully understood, we have investigated the effects of DTX on apoptosis and ras-->Erk-mediated signal transduction in human epidermoid KB, colon HT-29 and breast HCC1937 cancer cells. We have found that the exposure to 0.78 or 1.56 or 2.5 ng/ml DTX for 48 h induced apoptosis and growth inhibition in about 50 % of KB, HCC1937 and HT-29 cell population, respectively. In these experimental conditions, PARP and caspase 3 cleavage was also showed in all cell lines. KB and HCC1937 cells express a wild type p53 while HT-29 display a mutated form. Interestingly, we have found that DTX reduces the expression of mutated p53 in HT-29 and increases the expression of wild type in KB and HCC1937 cells. Moreover, DTX reduces ubiquitination of the wild type p53 in KB and HCC1937 cells and increases the ubiquitin-conjugated form of mutated p53 in HT-29 cells. Furthermore, exposure of cancer cells to DTX for 48 h increases the expression and activity of Ras and up-regulates Raf-1 and the phosphorylated isoforms of Erk-1/2. On the bases of these data, we have hypothesized that the increased activity of the ras-->erk-dependent pathway induced by DTX could be a protective signalling from the apoptosis caused by the drug. Therefore, we have used R115777, a farnesyl transferase inhibitor that inactivates ras, in combination with DTX. The combined treatment with DTX and R115777 resulted in a strong synergism in growth inhibition in the three cell lines. These data suggest the use of the combination in these therapeutic settings even if further experiments are required for the clinical translation.

Differential expression and cytoplasm/membrane distribution of endoglin (CD105) in human tumour cell lines: Implications in the modulation of cell proliferation.

Endoglin (CD105, an accessory component of the TGF-beta receptor complex) expression and distribution on different human tumour cells and its role in cellular proliferation were evaluated. We examined: 1) sixteen human carcinoma cell lines, 2) eight human sarcoma cell lines, 3) five miscellaneous tumour cell lines. HECV (endothelial cells) were employed as a positive control for endoglin expression. Normal Human Dermal Fibroblasts (NHDF) and 293 cells (epithelial kidney cells) were used as normal controls for connective and epithelial tissues, respectively. The results showed that CD105 was poorly expressed in the majority of human carcinoma cells (10/16), whereas it was highly expressed in most human sarcoma cells (7/8), and differently expressed by miscellaneous tumour cell lines. These data reflect endoglin expression by the normal counterparts of tumour cell lines, i.e. NHDF and 293 cells. However, CD105 levels in sarcoma cell lines, even though consistently lower than in NHDF, were significantly higher than those observed in carcinoma cells. Interestingly, CD105 presented a strong expression in the cytoplasm of MDA-MB-453 (breast carcinoma), NPA (papillary thyroid carcinoma), COLO-853 (melanoma) and SaOS-2 (osteosarcoma), but was weakly expressed on their cell membrane. This differential expression in the cytoplasm and on the membrane of some tumour cells, suggests a complex mechanism of translocation for this protein. The analysis of clonal growth in soft agar of some cell lines, characterized by high CD105 expression, showed an increased colony formation potential that was antagonized by the addition of anti-CD105 blocking mAb. The results indicated that endoglin is differentially expressed in human carcinoma and sarcoma cells and its overexpression modulates the proliferative rate of human solid tumour cells. Moreover, these data suggest that CD105 is involved in the regulation of TGF-beta effects in human solid malignancies, and therefore it could play an important role in tumour diagnosis and treatment.

Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells.

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.

The eukaryotic initiation factor 5A is involved in the regulation of proliferation and apoptosis induced by interferon-alpha and EGF in human cancer cells.

Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.