(-)-Epigallocatechin-3-gallate and hydroxytyrosol improved antioxidative and anti-inflammatory responses in bovine mammary epithelial cells.

(-)-Epigallocatechin-3-gallate (EGCG), the major phenolic compound of green tea, and hydroxytyrosol (HTyr), a phenol found in olive oil, have received attention due to their wide-ranging health benefits. To date, there are no studies that report their effect in bovine mammary gland. Therefore, the aim of this study was to evaluate the anti-oxidative and anti-inflammatory effects of EGCG and HTyr in bovine mammary epithelial cell line (BME-UV1) and to compare their antioxidant and anti-inflammatory in vitro efficacy. Sample of EGCG was obtained from a commercially available green tea extract while pure HTyr was synthetized in our laboratories. The mammary oxidative stress and inflammatory responses were assessed by measuring the oxidative stress biomarkers and the gene expression of inflammatory cytokines. To evaluate the cellular antioxidant response, glutathione (GSH/GSSH), γ-glutamylcysteine ligase activity, reactive oxygen species and malondialdehyde (MDA) production were measured after 48-h incubation of 50 µM EGCG or 50 µM of HTyr. Reactive oxygen species production after 3 h of hydrogen peroxide (50 µM H2O2) or lipopolysaccharide (20 µM LPS) exposure was quantified to evaluate and to compare the potential protection of EGCG and HTyr against H2O2-induced oxidative stress and LPS-induced inflammation. The anti-inflammatory activity of EGCG and HTyr was investigated by the evaluation of pro and anti-inflammatory interleukins (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10) messenger RNA abundance after treatment of cells for 3 h with 20 µM of LPS. Data were analyzed by one-way ANOVA. (-)-Epigallocatechin-3-gallate or HTyr treatments induced higher concentrations of intracellular GSH compared to control cells, matched by an increase of γ-glutamylcysteine ligase activity mainly in cells treated with HTyr. Interestingly, EGCG and HTyr prevented oxidative lipid damage in the BME-UV1 cells by a reduction of intracellular MDA levels. (-)-Epigallocatechin-3-gallate and HTyr were able to enhance cell resistance against H2O2-induced oxidative stress. It was found that EGCG and HTyr elicited a reduction of the three inflammatory cytokines TNF-α, IL-1ß, IL-6 and an increase of the anti-inflammatory cytokine IL-10. Hydroxytyrosol has proved to be a strong antioxidant compound, and EGCG has shown mainly an anti-inflammatory profile. These results indicated that EGCG and HTyr may provide dual protection because they were able to attenuate oxidative stress and inflammatory responses, suggesting that these phenolic compounds are potential natural alternatives to be used in dairy cattle as feed supplement for reducing the development of oxidative and inflammatory processes related to parturition or as topical treatments for the control of bovine intramammary inflammation.

Effect of hot season on blood parameters, fecal fermentative parameters, and occurrence of Clostridium tyrobutyricum spores in feces of lactating dairy cows.

High temperature influences rumen and gut health, passage rate, and diet digestibility, with effects on fermentative processes. The main aim of the study was to investigate the effect of hot season on hindgut fermentation, the occurrence of Clostridium tyrobutyricum spores in bovine feces, and on their relationship with metabolic conditions in dairy cows producing milk used for Grana Padano cheese. The study was carried out on 7 dairy farms located in the Po Valley (Italy), involving 1,950 Italian Friesian dairy cows. The study was carried out from November 2013 till the end of July 2014. Temperature and relative humidity were recorded daily by weather stations. Constant management conditions were maintained during the experimental period. Feed and diet characteristics, metabolic conditions, and fecal characteristics were recorded in winter (from late November 2013 to the end of January 2014), spring (from April to May 2014), and summer (July 2014) season. In each season, blood samples were collected from 14 multiparous lactating dairy cows per herd to measure biochemical indices related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. Fecal samples were also collected and measurements of moisture, pH and volatile fatty acids (VFA) were performed. The DNA extracted and purified from fecal samples was used to detect Clostridium tyrobutyricum spores in a quantitative real-time PCR assay. The daily mean temperature-humidity index was 40.7 ± 4.6 (range 25 to 55), 61.2 ± 3.7 (range 39 to 77), and 70.8 ± 3.2 (range 54 to 83) in winter, spring, and summer, respectively. Total VFA concentration in feces progressively decreased from winter to summer. The seasonal changes of acetate and propionate followed the same trend of total VFA; conversely, butyrate did not show any difference between seasons, and its molar proportion was greater in summer compared with winter. A greater occurrence of Cl. tyrobutyricum spores in summer compared with the other seasons was observed. The plasma concentrations of glucose, urea, albumin, Ca, Mg, Cl, Zn, and alkaline phosphatase activity were lower in summer compared with winter, whereas the opposite occurred for bilirubin and Na. Our results show that summer season, through direct and indirect effect of heat stress, affected fecal fermentative parameters and hindgut buffering capacity, and was responsible for the increasing occurrence of Cl. tyrobutyricum spores in feces.

Anti-inflammatory effects of conjugated linoleic acid isomers and essential fatty acids in bovine mammary epithelial cells.

Fatty acids are important modulators of inflammatory responses, in particular, n-3 and n-6 essential fatty acids and CLA have received particular attention for their ability to modulate inflammation. The objectives of this study were to compare the effects of CLA and essential fatty acids on the expression of pro and anti- inflammatory cytokines and their protective efficacy against inflammatory status in mammary gland by an in vitro model based on bovine mammary epithelial cells (BME-UV1). Bovine mammary epithelial cells were treated with complete medium containing either 50 µM of cis-9, trans-11 CLA (c9,t11 CLA) or trans-10, cis-12 CLA (t10,c12 CLA) or (α)-linolenic acid (aLnA) or (γ)-linolenic acid (gLnA) or linoleic acid (LA). After 48 h by fatty acids administration the cells were treated for 3 h with 20 µM of lipopolysaccharide (LPS) to induce inflammatory stimulus. Reactive oxygen species (ROS) production after treatments was assessed to verify and to compare the potential protection of different fatty acids against LPS-induced oxidative stress. The messenger RNA abundance of bovine pro and anti-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interleukine-10 (IL-10)) and peroxisome proliferator receptor-α/γ (PPARγ/α) were determined in BME-UV1 by real-time PCR. The results showed that cells treated with fatty acids and LPS increased ROS production compared with control cells. Among treatments, cells treated with c9,t11 CLA and t10,c12 CLA isomers revealed significant lower levels of ROS production compared with other fatty acids. All fatty acids reduced the gene expression of pro- and anti-inflammatory cytokines. Among fatty acids, t10,c12 CLA, LA and gLnA showed an homogeneous reduction of the three pro-inflammatory cytokines and this may correspond to more balanced and efficient physiological activity and may trigger a better protective effect. The PPARγ gene expression was significantly greater in cells treated with t10,c12 CLA, aLnA and LA, whereas the PPARα gene expression levels were significantly lower in cells treated with all different fatty acids, compared with the control. These results suggest that fatty acids inhibited the transcription of pro-inflammatory cytokines by the upregulation of PPARγ expression.

Comparison between conjugated linoleic acid and essential fatty acids in preventing oxidative stress in bovine mammary epithelial cells.

Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 µM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of H2O2 exposure was assessed to evaluate and to compare the potential protection of different FA against H2O2-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by H2O2 compared with other FA.

Fate of transgenic deoxyribonucleic acid fragments in digesta and tissues of rabbits fed genetically modified soybean meal.

Numerous animal feeding studies have investigated the presence of DNA from transgenic plants in tissues from different animal species, but the data reported are sometimes controversial. The aim of this study was to investigate the presence of transgenic DNA (tDNA) in the digesta and tissues of a meat rabbit breed fed genetically modified (GM) soybean meal. Fifteen male New Zealand White rabbits were used for the experimental trial. Ten rabbits (treated group [TG]) were fed a mixed feed containing 10% GM soybean meal and 5 rabbits (control group [CG]) received a mixed feed containing conventional soybean meal, both from weaning (28 d of age) to slaughter (80 ± 3 d). Samples of blood, liver, kidney, heart, stomach, intestine (jejunum), lateral quadricep muscle, longissimus muscle, and perirenal adipose tissue were collected to assess the possible DNA transfer from GM feed to animal tissues. Samples of stomach contents and feces were also taken to study the degradability of ingested tDNA from feed in the digestive tract of rabbit. Moreover, samples of hair were collected to determine the possible environmental contamination from feed powders present on the farm. The DNA extraction was performed using specific genomic DNA kits. All samples were monitored, by using real-time PCR, for oligonucleotide primers and probes specific for the transgenic Roundup Ready soybean 40-3-2 and for the endogenous () gene. As an internal control of rabbit tissues, the presence of the () gene was used. In this study, no fragments of tDNA were detectable in tissue DNA samples of rabbits except in the extracted DNA from stomach digesta, feces, and hair of rabbits fed with GM soybean. Similar results were found for the reference gene, whereas the presence of the gene was detected in all rabbit tissues. The lack of tDNA of soybean in rabbit tissues represents an important result, which demonstrates that meat from rabbits fed a diet containing GM feed is as that derived from rabbits fed conventional crops. The recombinant DNA recovered in the stomach digesta and in feces indicates an incomplete digestion of the soybean DNA in the gastrointestinal tract of the rabbit, whereas the presence of trace soybean transgene in the hair of the TG rabbits is suggestive of an environmental contamination.

Release of angiotensin converting enzyme-inhibitor peptides during in vitro gastrointestinal digestion of Parmigiano Reggiano PDO cheese and their absorption through an in vitro model of intestinal epithelium.

The occurrence of 8 bovine casein-derived peptides (VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP, and HLPLP) reported as angiotensin converting enzyme-inhibitors (ACE-I) was investigated in the 3-kDa ultrafiltered water-soluble extract (WSE) of Parmigiano Reggiano (PR) cheese samples by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry via an electrospray ionization source. Only VPP, IPP, LHLPLP, and HLPLP were revealed in the WSE, and their total amount was in the range of 8.46 to 21.55 mg/kg of cheese. Following in vitro static gastrointestinal digestion, the same ACE-I peptides along with the newly formed AYFYPEL and AYFYPE were found in the 3 kDa WSE of PR digestates. Digestates presented high amounts (1,880-3,053 mg/kg) of LHLPLP, whereas the remaining peptides accounted for 69.24 to 82.82 mg/kg. The half-maximal inhibitory concentration (IC50) values decreased from 7.92 ± 2.08 in undigested cheese to 3.20 ± 1.69 after in vitro gastrointestinal digestion. The 3-kDa WSE of digested cheeses were used to study the transport of the 8 ACE-I peptides across the monolayers of the Caco-2 cell culture grown on a semipermeable membrane of the transwells. After 1h of incubation, 649.20 ± 148.85 mg/kg of LHLPLP remained in the apical compartment, whereas VPP, IPP, AYFYPEL, AYFYPE, and HLPLP accounted in total for less than 36.78 mg/kg. On average, 0.6% of LHLPLP initially present in the digestates added to the apical compartment were transported intact to the basolateral chamber after the same incubation time. Higher transport rate (2.9%) was ascertained for the peptide HLPLP. No other intact ACE-I peptides were revealed in the basolateral compartment. For the first time, these results demonstrated that the ACE-I peptides HLPLP and LHLPLP present in the in vitro digestates of PR cheese are partially absorbed through an in vitro model of human intestinal epithelium.

Conjugated linoleic acid isomers strongly improve the redox status of bovine mammary epithelial cells (BME-UV1).

Some studies have shown the protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation in animal models, but no information is available about CLA and changes in oxidative status of the bovine mammary gland. The objectives of the study were to assess in vitro the effect of CLA on the cellular antioxidant response of bovine mammary cells, to examine whether CLA isomers could play a role in cell protection against the oxidative stress, and to study the molecular mechanism involved. For the study, BME-UV1 cells, a bovine mammary epithelial cell line, were used as the experimental model. The BME-UV1 cells were treated with complete medium containing 50 µM cis-9,trans-11 CLA (c9,t11 CLA), trans-10,cis-12 CLA (t10,c12 CLA), and CLA mixture (1:1, cis-9,trans-11: trans-10,cis-12 CLA). To monitor cellular uptake of CLA isomers, cells and culture medium were collected at 0, 3, and 48 h from CLA addition for lipid extraction and fatty acid analyses. To assess the cellular antioxidant response, glutathione (GSH/GSSH), NADPH, and γ-glutamyl-cysteine ligase activity was measured after 48 h from addition of CLA. Cytoplasmic superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase activities and mRNA were also determined. Intracellular reactive oxygen species and thiobarbituric acid reactive substance production were assessed in cells supplemented with CLA isomers. Cell viability after 3h to H2O2 exposure was assessed to evaluate and to compare the potential protection of different CLA isomers against H2O2-induced oxidative stress. Mammary cells readily picked up all CLA isomers, their accumulation was time dependent, and main metabolites at 48 h are two 18:3 isomers. The CLA treatment induced an intracellular GSH increase, matched by high concentration of NADPH, and an increase of γ-glutamyl-cysteine ligase activity mainly in cells treated with the t10,c12 CLA isomer. The CLA isomer treatment of bovine mammary cells increased superoxide dismutase, glutathione peroxidase, and glutathione S-transferase activity and decreased glutathione reductase activity, but no changes in gene expression of these antioxidant enzymes were observed. Cells supplemented with CLA isomers showed a reduction in intracellular reactive oxygen species and thiobarbituric acid reactive substance levels. All CLA isomers were able to enhance cell resistance against H2O2-induced oxidative stress. These suggest an antioxidant role of CLA, in particular of t10,c12 CLA, by developing a significantly high redox status in cells.

The effect of calving in the summer on the hepatic transcriptome of Holstein cows during the peripartal period.

The liver is the main metabolic organ coordinating the adaptations that take place during the peripartal period of dairy cows. A successful transition into lactation, rather than management practices alone, depends on environmental factors such as temperature, season of parturition, and photoperiod. Therefore, we analyzed the effect of calving season on the hepatic transcriptome of dairy cows during the transition period. A total of 12 Holstein dairy cows were assigned into 2 groups based on calving season (6 cows March-April, spring; 6 cows June-July, summer, SU). The RNA was extracted from liver samples obtained at -30, 3, and 35 DIM via percutaneous biopsy and hybridized to the Agilent 44K Bovine (V2) Gene Expression Microarray (Agilent Technologies Inc., Santa Clara, CA). A quantitative PCR on 22 target genes was performed to verify and expand the analyses. A total of 4,307 differentially expressed genes were detected (false discovery rate ≤0.05) in SU compared with spring. Furthermore, 73 unique differentially expressed genes were detected in SU compared with spring cows after applying a fold-change threshold ≥3 and ≤-3. For Kyoto Encyclopedia of Genes and Genomes pathways analysis of differentially expressed genes, we used the dynamic impact approach. Ingenuity Pathway Analysis software was used to analyze upstream transcription regulators and perform gene network analysis. Among metabolic pathways, energy metabolism from lipids, carbohydrates, and amino acids was strongly affected by calving in SU, with a reduced level of fatty acid synthesis, oxidation, re-esterification, and synthesis of lipoproteins, leading to hepatic lipidosis. Glycan-synthesis was downregulated in SU cows probably as a mechanism to counteract the progression of this lipidosis. In contrast, calving in the SU resulted in upregulation of gluconeogenesis but also greater use of glucose as an energy source. Among nonmetabolic pathways, the heat-shock response was obviously activated in SU cows but was also associated with inflammatory and intracellular stress response. Furthermore, data support a recent finding that cows experience endoplasmic reticulum stress around parturition. Transcription regulator analysis revealed how metabolic changes are related to important regulatory mechanisms, including epigenetic modification. The holistic analyses of the liver transcriptome response to calving in the summer at high environmental temperatures underscore how transition cows should be carefully managed during this period, as they experience alterations in liver energy metabolism and inflammatory state increasing susceptibility to health disorders in early postpartum.

Effect of summer season on milk protein fractions in Holstein cows.

Milk characteristics are affected by heat stress, but very little information is available on changes of milk protein fractions and their relationship with cheesemaking properties of milk. The main objective of the study was to evaluate the effect of hot season on milk protein fractions and cheesemaking properties of milk for Grana Padano cheese production. The study was carried out in a dairy farm with a cheese factory for transforming the milk to Grana Padano cheese. The study was carried out from June 2012 to May 2013. Temperature and relative humidity of the inside barn were recorded daily during the study period using 8 electronic data loggers programmed to record every 30 min. Constant managerial conditions were maintained during the experimental periods. During the experimental period, feed and diet characteristics, milk yield, and milk characteristics were recorded in summer (from June 29 to July 27, 2012), winter (from January 25 to March 8, 2013), and spring (from May 17 to May 31, 2013). Milk yield was recorded and individual milk samples were taken from 25 cows selected in each season during the p.m. milking. Content of fat, proteins, caseins (CN), lactose and somatic cell count (SCC), titratable acidity, and milk rennet coagulation properties were determined on fresh samples. Milk protein fraction concentrations were determined by the sodium dodecyl sulfate-PAGE. Data were tested for nonnormality by the Shapiro-Wilk test. In case of nonnormality, parameters were normalized by log or exponential transformation. The data were analyzed with repeated measures ANOVA using a mixed model procedure. For all the main milk components (fat, protein, total solids, and solids-not-fat), the lowest values were observed in the summer and the greatest values were observed in the winter. Casein fractions, with the exception of γ-CN, showed the lowest values in the summer and the greatest values in the winter. The content of IgG and serum albumin was greater in summer than in the winter and spring. A mild effect of season was observed for milk SCC, with greater values in summer than in the winter and spring. A worsening of milk coagulation properties was observed in summer season. The alteration of cheesemaking properties during hot season seems strictly linked with changes of milk protein fractions mainly with the decrease of αS-CN and ß-CN and the increase of undefined proteins.

Impact of hot environment on colostrum and milk composition.

Under hot and warm environments productivity and reproduction efficiency of farm and wild animals are negatively affected. The negative effects of hot environments on animal health are responsible for the alteration of colostrum and milk production in term of quantity and quality. Colostrum and milk are nutrient-rich fluids secreted by the mammary gland of female mammals after giving birth and during growth and development of the young. Multiple factors influence the production and the composition of colostrum and milk, including species, breed, health status, feeding practices and environmental conditions. Colostrum and milk are not only a good source of macronutrients and micronutrients, but contains many biologically-active constituents. Colostrum and milk of various species differ widely in amounts and proportions of their principal constituents, especially comparing monogastric with ruminant animals because of the difference between their physiology and digestion. The interspecies variations in part reflect different adaptive strategies to environmental conditions and selective pressures of various species during the evolution. A limited number of studies documented the effects of hot condition on modification of colostrum and milk quality, in particular referred to nutrients and immunoglobulin composition, but no information are available on the effects of hot environment on nutraceutical properties and bioactive molecules content of colostrum and milk.

Moving towards high-power, high-frequency and low-resistance CNT supercapacitors by tuning the CNT length, axial deformation and contact resistance.

In this paper it is shown that the electrochemical behaviour of vertically aligned multi-walled carbon nanotube (VANT) supercapacitors is influenced by the VANTs' length (electrode thickness), by their axial compression and by their interface with the current collector. It is found that the VANTs, which can be interpreted as a dense array of nanochannels, have an active area available to ions that is strongly affected by the electrode's thickness and compressional state. Consequently, the tested thinner electrodes, compressed electrodes or a combination of the two were found to be characterized by a significant improvement in terms of power density (up to 1246%), knee frequency (58,822% working up to 10 kHz), equivalent series resistance (ESR, up to 67%) and capacitance (up to 21%) when compared with thicker and/or uncompressed electrodes. These values are significantly higher than those reported in the literature where long VANTs with no control on compression are typically used. It is also shown that the ESR can be reduced not only by using shorter and compressed VANTs that have a higher conductance or by improving the electrode/collector electrical contact by changing the contact morphology at the nanoscale through compression, but also by depositing a thin platinum layer on the VANT tips in contact with the current collector (73% ESR decrease).

Heat shock impairs DNA synthesis and down-regulates gene expression for leptin and Ob-Rb receptor in concanavalin A-stimulated bovine peripheral blood mononuclear cells.

This study verified whether leptin or its long isoform receptor (Ob-Rb) genes are expressed in proliferating lymphocytes from bovine species, and whether their expression changes with increased temperatures. Peripheral blood mononuclear cells (PBMC) from five Holstein cows were incubated in the presence of concanavalin A, and alternatively subjected for 65 h to each of the following treatments (T): 39 degrees C continuously (T39) or three 13-h cycles at 40 (T40), 41 (T41) or 42 degrees C (T42), respectively, which were alternated with two 13-h cycles at 39 degrees C. T39 mimicked normothermia; T40, 41 and 42 mimicked conditions of hyperthermia alternated with normothermia. PBMC proliferation declined under T42. Compared with T39, levels of mRNA for leptin was lower under T42, whereas mRNA for Ob-Rb was lower in lymphocytes cultured both under T41 and T42. DNA synthesis was positively correlated with leptin mRNA. This study supports the concept that severe heat stress impairs proliferation of bovine PBMC, confirms that bovine lymphocytes express Ob-Rb gene, and provides the first experimental evidence that bovine lymphocytes express gene for leptin, and that increased temperatures are associated with altered gene expression for leptin and Ob-Rb.

Photoperiod affects gene expression of leptin and leptin receptors in adipose tissue from lactating dairy cows.

Leptin is mainly secreted by adipocytes and is implicated in the regulation of metabolic status, feed intake, and body condition. Day length (DL) can affect leptin gene expression and secretion. The aim of the study was to evaluate the effect of DL on gene expression of leptin and leptin receptors in adipose tissue (AT). Four lactating and pregnant Holstein cows were housed in a climate-controlled chamber for 51 d. The first 30 d were used to adapt animals to the new housing conditions. During that period the DL adopted was 12 h light:12 h dark (12:12). The experimental period included 3 different and consecutive phases: 7 d of neutral DL (12:12); 7 d of long DL (18 h light:6 h dark); and 7 d of short DL (6 h light:18 h dark). Subcutaneous AT biopsies were performed at the end of each phase. Prolactin, growth hormone, cortisol, leptin, glucose, nonesterified fatty acids, beta-OH-butyrate, and cholesterol were determined in plasma samples. Abundance of leptin mRNA, and Ob-Ra and Ob-Rb leptin receptor mRNA were determined in AT samples by ribonuclease protection assay. Day length did not affect feed intake or body condition score. Exposure to short DL significantly reduced milk yield (13.1 +/- 2.2 vs. 15.8 +/- 1.7 and 16.0 +/- 2.0 kg/d for short vs. neutral and long DL, respectively). Plasma leptin, growth hormone, cortisol, nonesterified fatty acids, beta-OH-butyrate, and glucose were not affected by DL; cholesterol was lowest under short DL (3.93 +/- 0.38 vs. 4.36 +/- 0.39 and 4.07 +/- 0.38 mmol/L for short vs. neutral and long DL, respectively). Prolactin increased under long DL (134.82 +/- 16.94 vs. 81.98 +/- 20.25 and 96.16 +/- 0.38 ng/mL for long vs. neutral and short DL, respectively). Gene expression of leptin and its receptors was affected by DL. Leptin mRNA increased under long DL (11.91 +/- 0.84 vs. 7.82 +/- 0.84 and 7.56 +/- 0.84 pg of mRNA/microg of total RNA for long vs. neutral and short DL, respectively). Leptin receptors Ob-Ra and Ob-Rb mRNA were higher under long DL, whereas Ob-Ra and Ob-Rb mRNA were lower under short DL (Ob-Ra: 1.91 +/- 0.41, 2.49 +/- 0.41, and 0.65 +/- 0.41 pg of mRNA/microg of total RNA for neutral, long, and short DL, respectively; Ob-Rb: 5.29 +/- 0.79, 5.98 +/- 0.68, and 2.02 +/- 0.70 pg of mRNA/microg of total RNA for neutral, long, and short DL, respectively). Results of the present study appear to exclude an effect of feed intake and metabolic status on leptin gene expression. A prolactin-mediated effect of photoperiod on AT leptin modulation may be proposed in lactating dairy cows.

Heat stress elicits different responses in peripheral blood mononuclear cells from Brown Swiss and Holstein cows.

This study was undertaken to assess whether peripheral blood mononuclear cells (PBMC) isolated from Brown Swiss (Br) and Holstein (Ho) cows and stimulated with concanavalin A differ in response to chronic exposure to incubation temperatures simulating conditions of hyperthermia. Five multiparous Br and 5 Ho cows were utilized as blood donors. Peripheral blood mononuclear cells were subjected for 65 h to each of 5 treatments (T). Cells were exposed to 39 degrees C continuously (T39) and three 13-h cycles at 40 (T40), 41 (T41), 42 (T42) or 43 degrees C (T43), respectively, which were interspersed with two 13-h cycles at 39 degrees C. Treatment T39 was adopted to mimic normothermia; T40, T41, T42, and T43 mimicked conditions of more severe hyperthermia alternating with normothermia. Measures evaluated at the end of the incubation period were proliferative response (DNA synthesis), intracellular reactive oxygen species (ROS) concentrations, and mRNA abundance of the 72-kDa heat-shock protein (Hsp72). In Br cows, DNA synthesis began to decline when PBMC were repeatedly exposed to 41 degrees C (-22%), whereas DNA synthesis in cells isolated from Ho cows did not begin to decline until 42 degrees C (-40%). Furthermore, under T41 and T42, DNA synthesis from Br cows was lower than in Ho(-24 and -54%, respectively). In both breeds, increased incubation temperatures caused a reduction of intracellular ROS (from -39.6 and -69.7%). Increase in incubation temperatures enhanced Hsp72 mRNA levels only in PBMC isolated from Br cows. The Hsp72 mRNA in Br cows increased significantly under T41 and T43 compared with T39. In both breeds, DNA synthesis was positively and negatively correlated with intracellular ROS and Hsp72 mRNA abundance, respectively (r = 0.85 and r = -0.70, respectively). Results indicated that PBMC from Br cows are less tolerant to chronic heat exposure than those from Ho cows, and that the lower tolerance is associated with higher expression of Hsp72, suggesting that the same level of hyperthermia may be associated with a differential decline of immune function in the 2 breeds.

Abundance of mRNA of apolipoprotein b100, apolipoprotein e, and microsomal triglyceride transfer protein in liver from periparturient dairy cows.

Limited secretion of very low density lipoproteins (VLDL) in dairy cows is strongly related to fatty liver and other metabolic disorders in the early postpartum. Currently, there is limited information on which roles apolipoprotein B(100) (ApoB(100)), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTP) play in that VLDL limitation. To our knowledge, no studies have simultaneously measured ApoB(100), ApoE, and MTP mRNA in periparturient dairy cows. Therefore, a trial was conducted to assess liver gene expression of these proteins in transition dairy cows and to evaluate the relationships between their expression and metabolic status. Eight multiparous Holstein cows were monitored during the transition period. To evaluate metabolic and nutritional status, body condition score was registered, and plasma indexes of energy metabolism and VLDL were determined from 35 d before to 35 d after calving. Liver biopsies were performed on d -35, 3, and 35 relative to day of calving, and gene expression of ApoB(100), ApoE, and MTP were determined on liver tissue. Body condition, plasma glucose and VLDL decreased, and plasma NEFA and BHBA increased after calving. Compared with values of d -35, on d 3 after calving the ApoB(100) mRNA synthesis was lower, whereas MTP and ApoE mRNA abundance were higher. Negative correlation (r = -0.57) between plasma NEFA concentration and ApoB(100) mRNA abundance, and positive correlation between ApoB(100) mRNA abundance and plasma cholesterol (r = 0.65) and plasma albumins (r = 0.52) were detected at 3 d postpartum. Data on changes of gene expression of the 3 main proteins involved in the regulation of synthesis and secretion of VLDL in the liver suggest that decreased mRNA for ApoB(100) may be consistent with decreased synthesis and/or secretion of VLDL from liver during the periparturient period.

Absence of regular alpha2(I) collagen chains in colon carcinoma biopsy fragments.

The extracellular matrix (ECM) is known to play an active role in numerous biological processes such as differentiation, apoptosis and cancer. Extensive alterations of epithelial basement membranes and of interstitial ECM are known to occur during the progression of most invasive carcinomas. Collagen, which represents the major component of the interstitial ECM, is primarily involved in the stromal changes at the site of tumor cell invasion. We have previously described the occurrence in breast and colon cancer ECM of an oncofetal form of collagen, characterized by an acidic chain distinct from those of type I and III collagen. In the present paper, we bring evidence that alpha2(I) collagen chains in colon cancer tissues expressing the acidic chains, are either overmodified or absent, both as protein and as regular mRNA transcripts. The results obtained strongly suggest that: i) the disorganisation of the collagen architecture and the phenomenon of fibril dispersion, which accompanies the lysis of basement membrane, is not only due to the enzymatic degradation of the collagen fibres, but presumably also to changes of the collagen molecules deposited in the stroma; ii) the neosynthesis of collagen occurring at tumor-host interface is deeply deregulated, and therefore to be considered the result of altered collagen gene expression correlated with the tumor progression, rather than as a mere defensive reaction of the host cells.

Onco-fetal/laminin-binding collagen from colon carcinoma: detection of new sequences.

We have recently identified an oncofetal-laminin binding collagen (OF/LB) composed of three alpha chains, with the apparent molecular mass of about 100 kDa each, but bearing different pI. One of the chains appears markedly acidic in a bidimensional electrophoretic system, where the NEPHGE is used as first dimension separating gel, while the two more basic chains have similar migration as alpha 1(III) and alpha 1(I) collagen chains, respectively. Sequence analyses have been performed on CNBr-peptides, derived from pepsinized triple helical molecules and on tryptic fragments obtained after in gel digestion of the acidic band. The research of sequence homology with computerized databases indicated that the acidic chain represents a gene product distinct from either type I, type III and other known collagen chains, while the identity of the other two chains remains to be fully determined.

Of/Lb collagen promotes chemoinvasion of breast-cancer cells and directs epithelial-cell migration into granulation-tissue of experimental dermal wounds.

OF/LB collagen is a recently described once-fetal form of collagen, with laminin-binding properties, composed of three alpha(1)(I)-sized chains, one of which displaying an unusually acidic pI. This collagen appears able to direct the migration of breast cancer cells through Matrigel, and of injury-activated epithelial cells into the underlying granulation stromal tissue. The effect exerted by OF/LB collagen in vitro appears preferentially linked to its acidic chain. The data reported strongly support the hypothesis that the presence and accumulation of OF/LB collagen in cancer may play a fundamental role in the invasive growth.

A new form of tumor and fetal collagen that binds laminin.

Human breast and colon carcinoma tissues contain a form of collagen, not described before, composed of alpha 1 chains of similar size (approximately 100 kDa) but different charge. The three constitutive chains, separated by two-dimensional electrophoresis, are a unique acidic component, undetectable in other collagen types, with an apparent isoelectric point of 4-5, and two more basic components displaying the same electrophoretic behavior as alpha 1(III) and alpha 1(I), respectively. The acidic chain is structurally distinct from alpha 1(I) and displays a cyanogen bromide-derived fragment of similar size to CB5(III). This collagen in its native state is resistant to trypsin and metalloproteinase 3, while it is fully degraded by metalloproteinases 1 and 9. Moreover, this collagen appears able to bind to laminin, as tested by affinity chromatography. The biological significance of our data is related to the finding of this collagen form not only in the tumor tissue tested but also in embryonic-fetal tissues (bovine skin and intestine and human umbilical cord). For its peculiar laminin-binding ability and occurrence in tumoral and embryonic-fetal tissues, we propose to temporarily term this new collagen form OF/LB collagen (onco-fetal, laminin-binding collagen). The presence of OF/LB collagen during development and cancer, and its absence in normal adult tissues, make this protein a potential stromal marker of malignancy.