Short communication: Circulating fatty acylcarnitines are elevated in overweight periparturient dairy cows in association with sphingolipid biomarkers of insulin resistance

Defects in mitochondrial fatty acid processing are associated with the development of fatty liver disease, inflammation, and insulin resistance in overweight nonruminants. Surplus fatty acids (FA) and defects in FA oxidation favor the accumulation of fatty acylcarnitines (FAC) and the sphingolipid ceramide. Moreover, elevated circulating FAC and ceramide concentrations are inversely related to insulin action. Because we have previously demonstrated that plasma ceramide levels increase during the transition from gestation to lactation, our aim was to determine whether changes in plasma medium- and long-chain FAC levels are related to circulating FA and sphingolipids in peripartal dairy cows. We hypothesized that plasma FAC levels would be higher in overweight cows experiencing increased lipolysis, and that FAC levels would be positively associated with elevations in plasma ceramides. Twenty-one multiparous Holstein cows were grouped according to body condition score (BCS) at d ?30 prepartum as lean (BCS <3.0; n = 10) or overweight (BCS >4.0; n = 11). Blood was collected at d ?30, ?15, ?7, and 4, relative to parturition. Circulating FAC and ceramide levels were determined using liquid chromatography and tandem mass spectrometry. To investigate the potential contributions of sphingomyelin (SM) hydrolysis to ceramide accrual, we also determined plasma SM levels during the peripartum period. Data were analyzed under a mixed model with the fixed effects of adiposity and time, and the random effect of cow. Relative to lean cows, overweight cows had elevated FAC during the transition from gestation to lactation. Circulating FAC levels were positively associated with FA, ceramide, and dihydro-SM levels. Although circulating FAC levels increased in all cows during the peripartum, enhanced prepartum adiposity contributed to a greater rise in plasma FA and FAC. Our results support on-going efforts to determine whether altered mitochondrial FA processing promotes the accumulation of the insulin resistance biomarker ceramide in blood and liver.     

Differentially expressed plasma microRNAs in premature ovarian failure patients and the potential regulatory function of mir-23a in granulosa cell apoptosis

Recent studies implicate the regulatory function of microRNAs (miRNAs) in oocyte maturation and ovarian follicular development. Differentially expressed miRNAs are found in the plasma of premature ovarian failure (POF) patients and normal cycling women. In this study, miRNA-regulated signaling pathways and related genes were described using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The effect of mir-23a on granulosa cell apoptosis was also studied by examining the protein expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3, followed by subsequent counting of apoptotic cells after Hoechst 33258 staining. Both GO analysis and pathway analysis suggested that many signaling pathways, including the AKT signaling pathway, steroid hormone receptor signaling pathways, and others, were regulated by this group of differentially expressed miRNAs. A decrease in XIAP expression (mRNA and protein level) and caspase-3 protein levels and an increase in cleaved caspase-3 protein were observed in human ovarian granulosa cells transfected with pre-mir-23a, along with an increased occurrence of apoptosis. In conclusion, differentially expressed miRNAs in the plasma of POF patients may have regulatory effects on proliferation and apoptosis of granulosa cells by affecting different signaling pathways. Mir-23a may play important roles in regulating apoptosis via decreasing XIAP expression in human ovarian granulosa cells.     

Events of elevated somatic cell counts in high-producing dairy cows are associated with daily body weight loss in early lactation

The objective of this study was to determine associations between body weight (BW) and body condition score (BCS) variables indicating a more severe negative energy balance in early lactation and events of somatic cell counts (SCC) >250,000 cells/mL and SCC >400,000 cells/mL in dairy cows. We studied lactations from 634 primiparous and 1,086 multiparous Israeli Holstein dairy cows originating from 7 commercial dairy farms. Generalized mixed models with a random herd effect were used to quantify the effects of BW and BCS variables in early lactation on events of elevated SCC. Data were analyzed using 2 different approaches. In the first approach, only first events in a lactation were taken into account, whereas in the second approach, all events in a lactation were analyzed and repeated events from the same cow were accounted for. Although no associations were found between the different BW and BCS variables and first events of elevated SCC, associations were present between these variables and events of elevated SCC when all events were analyzed. The cumulative incidence of a lactation with multiple events of SCC >250,000 cells/mL was 8.8 and 27.7% for primiparous and multiparous cows, respectively. The odds of an event of SCC >250,000 cells/mL were 25% greater for cows belonging to the upper quartile in relative BW loss from calving to nadir BW (loss >12.3, 15.0, and 15.7% for first-, second-, and third- parity and greater cows, respectively) compared with cows losing less relative BW. Odds of an event were 44% greater for cows with ketosis when compared with cows without. The cumulative incidence of a lactation with multiple events of SCC >400,000 cells/mL was 4.1 and 14.3% for primiparous and multiparous cows, respectively. The odds of an event of SCC >400,000 cells/mL were 43% greater for cows belonging to the upper quartile in relative BW loss from calving to nadir BW compared with cows losing less relative BW. Odds of an event were 33% greater for cows with ketosis when compared with cows without. Assuming that extreme BW loss and ketosis in early lactation indicate a more severe negative energy balance, our findings support the hypothesis that greater negative energy balance in early lactation predisposes dairy cows to udder inflammation. Considering the fact that many of the events were recurring, we stress the importance of including all events in the analysis and postulate the possibility of long-term detrimental effects of negative energy balance on udder health.     

海水循环冷却系统排放水浓缩规律及制盐研究

文章研究了海水循环冷却系统排放浓海水在室内自然蒸发过程中,CaSO4、NaCl的析出规律、主要离子的浓度变化规律以及日晒盐的化学组成和毒性.试验结果表明:海水循环冷却系统排放浓海水的CaSO4析出点为13.89°Be′,NaCl析出点为25.76°Be′,此时CaSO4的析出率为90.32％;实验制得的日晒盐化学组成符合制盐业一级品标准,并属实际无毒.因此,利用海水循环冷却系统排放浓海水制盐切实可行.     

Milk fatty acids as possible biomarkers to early diagnose elevated concentrations of blood plasma nonesterified fatty acids in dairy cows

Most cows encounter a state of negative energy balance during the periparturient period, which may lead to metabolic disorders and impaired fertility. The aim of this study was to assess the potential of milk fatty acids as diagnostic tools of detrimental levels of blood plasma nonesterified fatty acids (NEFA), defined as NEFA concentrations beyond 0.6 mmol/L, in a data set of 92 early lactating cows fed a glucogenic or lipogenic diet and subjected to 0-, 30-, or 60-d dry period before parturition. Milk was collected in wk 2, 3, 4, and 8 (n = 368) and blood was sampled weekly from wk 2 to 8 after parturition. Milk was analyzed for milk fatty acids and blood plasma for NEFA. Data were classified as “at risk of detrimental blood plasma NEFA” (NEFA ≥0.6 mmol/L) and “not at risk of detrimental blood plasma NEFA” (NEFA <0.6 mmol/L). Concentrations of 45 milk fatty acids and milk fat C18:1 cis-9-to-C15:0 ratio were subjected to a discriminant analysis. Milk fat C18:1 cis-9 revealed the most discriminating variable to identify detrimental blood plasma NEFA. A false positive rate of 10% allowed us to diagnose 46% of the detrimental blood plasma NEFA cases based on a milk fat C18:1 cis-9 concentration of at least 230 g/kg of milk fatty acids. Additionally, it was assessed whether the milk fat C18:1 cis-9 concentrations of wk 2 could be used as an early warning for detrimental blood plasma NEFA risk during the first 8 wk in lactation. Cows with at least 240 g/kg of C18:1 cis-9 in milk fat had about 50% chance to encounter blood plasma NEFA values of 0.6 mmol/L or more during the first 8 wk of lactation, with a false positive rate of 11.4%. Profit simulations were based on costs for cows suffering from detrimental blood plasma NEFA, and costs for preventive treatment based on daily dosing of propylene glycol for 3 wk. Given the relatively low incidence rate (8% of all observations), continuous monitoring of milk fatty acids during the first 8 wk of lactation to diagnose detrimental blood plasma NEFA does not seem cost effective. On the contrary, milk fat C18:1 cis-9 of the second lactation week could be an early warning of cows at risk of detrimental blood NEFA. In this case, selective treatment may be cost effective.     

Circulating low-density lipoprotein ceramide concentrations increase in Holstein dairy cows transitioning from gestation to lactation

Low-density lipoprotein (LDL) ceramide causes insulin resistance in obese diabetic nonruminants. Because previous work suggests that liver-derived ceramide may impair insulin action in postpartum cows, our objectives were to characterize peripartal changes in lipoprotein ceramides. We further studied the effects of prepartum adiposity on lipoprotein ceramide levels. Twenty-eight pregnant Holstein cows (parity = 3.65 ± 1.62) with lean (body condition score, BCS = 2.97 ± 0.16; body weight, BW = 630 ± 55.2 kg; n = 15) or overweight (BCS = 3.93 ± 0.27; BW = 766 ± 46.1 kg; n = 13) body condition 28 d before expected parturition were evaluated. Sampling occurred on d ?20.5 ± 1.74, ?13.8 ± 1.71, ?7.84 ± 4.07, ?6.71 ± 1.00, ?3.92 ± 0.64, and ?1.28 ± 0.61 (before parturition); daily until d 8 postpartum; and on d 10, 12, 14, 21, and 28. Adipose tissue and liver were biopsied on d ?7.84 ± 4.07 and 10. Postpartum insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp. Lipoprotein fractions were isolated using liquid chromatography. Sphingolipids were quantified using mass spectrometry. Data were analyzed using a mixed model with repeated measures. Overweight cows had a higher BCS and BW at enrollment relative to lean cows, but BCS and BW were similar postpartum. Overweight cows lost more body condition (0.97 ± 0.36 vs. 0.55 ± 0.16 BCS units) and BW (291 ± 67.3 vs. 202 ± 54.5 kg) during transition relative to lean cows. Adipocyte volume and counts declined from prepartum to postpartum (50.4 and 13.7%, respectively), and adipocyte volume was greater (48.2%) in overweight cows prepartum relative to lean cows. Although DMI was comparable between BCS groups, milk yield tended to be greater in overweight cows. Plasma free fatty acid and β-hydroxybutyrate and liver lipid levels were 40, 16, and 37% greater, respectively, in overweight cows compared with lean cows. Glucose infusion rate during the hyperinsulinemic-euglycemic clamp tended to be lower in overweight cows. Ceramide levels within triacylglycerol-rich lipoprotein fractions declined postpartum, whereas LDL ceramide increased postpartum. Overweight cows had lower triacylglycerol-rich lipoprotein C16:0-ceramide levels relative to lean cows. Prepartum LDL C24:0-ceramide levels were greater in overweight cows relative to lean cows. Independent of prepartum adiposity, we concluded that serum LDL ceramide levels are elevated in early-lactation cows experiencing adipose tissue free fatty acid mobilization and hepatic steatosis.     

Short communication: estimation of plasma osmolality from plasma electrolytes, glucose, and urea in calves

Plasma osmolality estimated from plasma concentrations of Na+, Cl-, K+, glucose, and urea was compared with measured osmolality in preweaned Holstein calves. When calves (n = 5) were fed only milk replacer after fasting for 24 h, measured osmolality fluctuated almost in parallel with estimated osmolality during the 8-h period after feeding, although estimated values were about 90% of measured values. When calves (n = 5) were fed only calf starter after fasting for more than 16 h, measured osmolality did not parallel the estimated osmolality during the 8-h period after feeding. Some factors depressed measured osmolality in the first 2 h.     

Lipodepsipeptides from Pseudomonas syringae are partially proteolyzed and are not absorbed by humans: an in vitro study

There are some concerns about the use of Pseudomonas-based products as biocontrol agents because of the hemolytic activity shown by their metabolites. The effects of Pseudomonas lipodepsipeptides (LDPs) on mammals via ingestion and the LDP degradation during the digestion and intestinal permeability have not been evaluated. In this research, the susceptibility of different LDPs to degradation was assayed with enzymatic gastrointestinal digestion, and intestinal permeability to LDPs was investigated in an in vitro system based on an intestinal cell layer system. Results demonstrated that trypsin and chymotrypsin hydrolyze up to 50% of the various LDPs, and that proteolysis was further increased by pronase E treatment. A decrease in LDP hemolytic activity matched LDP degradation during the various steps of the digestion process. Moreover, it was shown that syringomycin E (SRE), the main known LDP, was not able to cross the intestinal cell layer, suggesting that SRE does not reach the bloodstream in vivo. It was concluded that the Pseudomonas-based biocontrol products do not represent a serious risk for consumer health. In fact, LDPs possibly present on biocontrol-treated agricultural commodities would likely be partially digested by gastrointestinal enzymes and would not be absorbed at the intestinal level.     

Slower proteolysis in Cheddar cheese made from high-protein cheese milk is due to an elevated whey protein content

A growing number of companies within the cheese-making industry are now using high-protein (e.g., 4–5%) milks to increase cheese yield. Previous studies have suggested that cheeses made from high-protein (both casein and whey protein; WP) milks may ripen more slowly; one suggested explanation is inhibition of residual rennet activity due to elevated WP levels. We explored the use of microfiltration (MF) to concentrate milk for cheese-making, as that would allow us to concentrate the casein while varying the WP content. Our objective was to determine if reducing the level of WP in concentrated cheese milk had any impact on cheese characteristics, including ripening, texture, and nutritional profile. Three types of 5% casein standardized and pasteurized cheese milks were prepared that had various casein:true protein (CN:TP) ratios: (a) control with CN:TP 83:100, (b) 35% WP reduced, 89:100 CN:TP, and (c) 70% WP reduced, 95:100 CN:TP. Standardized milks were preacidified to pH 6.2 with dilute lactic acid during cheese-making. Composition, proteolysis, textural, rheological, and sensory properties of cheeses were monitored over a 9-mo ripening period. The lactose, total solids, total protein, and WP contents in the 5% casein concentrated milks were reduced with increasing levels of WP removal. All milks had similar casein and total calcium levels. Cheeses had similar compositions, but, as expected, lower WP levels were observed in the cheeses where WP depletion by MF was performed on the cheese milks. Cheese yield and nitrogen recoveries were highest in cheese made with the 95:100 CN:TP milk. These enhanced recoveries were due to the higher fraction of nitrogen being casein-based solids. Microfiltration depletion of WP did not affect pH, sensory attributes, or insoluble calcium content of cheese. Proteolysis (the amount of pH 4.6 soluble nitrogen) was lower in control cheeses compared with WP-reduced cheeses. During ripening, the hardness values and the temperature of the crossover point, an indicator of the melting point of the cheese, were higher in the control cheese. It was thus likely that the higher residual WP content in the control cheese inhibited proteolysis during ripening, and the lower breakdown rate resulted in its higher hardness and melting point. There were no major differences in the concentrations of key nutrients with this WP depletion method. Cheese milk concentration by MF provides the benefit of more typical ripening rates.     

Specific negative effects resulting from elevated levels of the recombinational repair protein Rad54p in Saccharomyces cerevisiae

RAD54 is an important gene in the RAD52 group that controls recombinational repair of DNA damage in Saccharomyces cerevisiae. Rad54p is a DNA‐dependent ATPase and shares seven conserved sequence motifs with proteins of the Swi2p/Snf2p family. Genetic analysis of mutations in motif IA, the putative ATP‐binding fold of Rad54p, demonstrated the functional importance of this motif. Overexpression of these mutant proteins resulted in strong, dominant‐negative effects on cell survival. High levels of full‐length wild‐type Rad54p or specific parts of Rad54p also resulted in negative effects, dependent on the ploidy of the host cell. This differential effect was not under a /α mating‐type control. Deletion of the RAD54 gene led to a small but significant increase in the mutation rate. However, the negative overexpression effects in haploid cells could not be explained by an accumulation of (recessive) lethal mutations. All negative overexpression effects were found to be enhanced under genotoxic stress. We suggest that the negative overexpression effects are the result of unbalanced protein–protein interactions, indicating that Rad54p is involved in multiple interactions, dependent on the physiological situation. Diploid wild‐type cells contained an estimated 7000 Rad54p molecules/cell, whereas haploid cells about 3500/cell. Rad54p levels were highest in actively growing cells compared to stationary phase cells. Rad54 protein levels were found to be elevated after DNA damage. Copyright © 1999 John Wiley & Sons, Ltd.     

The yeast Rvs161 and Rvs167 proteins are involved in secretory vesicles targeting the plasma membrane and in cell integrity

The Rvs161 and Rvs167 proteins are known to play a role in actin cytokeleton organization and endocytosis. Moreover, Rvs167p functionally interacts with the myosin Myo2p. Therefore, we explored the involvement of the Rvs proteins in vesicle traffic and in cell integrity. The rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction. They are synthetic-lethal with the slt2/mpk1 mutation, which affects the MAP kinase cascade controlled by Pkc1p and is required for cell integrity. The phenotype of the double mutants is close to that described for the pkc1 mutant. Synthetic defects for growth are also observed with mutation in KRE6, a gene coding for a glucan synthase, required for cell wall construction. These data support the idea that the Rvs proteins are involved in the late targeting of vesicles whose cargoes are required for cell wall construction.     

Short communication: Prepartum plasma insulin-like growth factor-I concentrations based on day of insemination are lower in cows developing postpartum diseases

Because peripartal production diseases are prevalent in dairy cows, early recognition is crucial. Several studies reported metabolic variables as risk predictors for subsequent diseases. To improve on-farm testing and application of those methods, the sampling procedure should take into account variation in gestation length. Furthermore, additional variables indicating cows at risk of any production disease should be sought. Therefore, the objective was to characterize differences between cows with and without postpartum production disease (retained fetal membranes, ketosis, hypocalcemia, abomasal displacement, metritis, mastitis) by prepartum measurement of serum nonesterified fatty acid (NEFA) and plasma insulin-like growth factor (IGF)-I concentrations relative to the artificial insemination (AI) that established pregnancy. Blood was collected from 41 Holstein Friesian cows on 235 to 241, 242 to 248, 249 to 255, 256 to 262, 263 to 269, 270 to 276, 277 to 283, and 284 to 290 d after AI. Health status was assessed daily for 3 wk after calving; 25 cows (66%) had at least one production disease. Cows developing postpartum diseases had higher mean serum NEFA concentrations (450 ± 26 μmol/L; mean ± SE) and lower plasma IGF-I concentrations (78 ± 6 ng/mL) prepartum compared with healthy cows (259 ± 19 μmol/L and 117 ± 8 ng/mL, respectively). In conclusion, because of substantial variation among cows in gestation length, blood samples should be collected and studies performed on risk prediction relative to AI rather than expected date of calving. As the somatotropic axis is one of the key regulators of metabolic adaption for onset of lactation, IGF-I might be a useful variable to differentiate between cows susceptible to production diseases and cows that are able to adapt adequately within the transition period and remain healthy.     

柱层析法提取刺玫果肉活性成分及其体外活性

探究柱层析循环联合法提取刺玫果肉中目标成分(多糖、总黄酮、总皂苷)的工艺条件及其体外活性。以提取液中目标成分的含量为指标,通过对乙醇体积分数、吸涨体积、浸泡平衡时间、超声时间的考察确定最佳提取工艺。测定刺玫果肉目标成分的提取物对α-葡萄糖苷酶的抑制率及对亚硝酸盐的清除率。结果表明,在最佳提取条件下,刺玫果肉目标成分的提取率均在90%以上;分离后,干浸膏中多糖、总黄酮、总皂苷的含量分别为428.5、484.5、122.5 mg/g。纯化后,干浸膏中多糖、总黄酮、总皂苷的含量分别为804.1、772.7、396.4 mg/g。刺玫果肉目标成分的提取物抑制α-葡萄糖苷酶活性的能力均强于阳性对照阿卡波糖,表明其具有显著的体外降糖作用;刺玫果肉目标成分的提取物清除亚硝酸盐的IC₅₀值均高于阳性对照维生素C,表明其具有较弱的亚硝酸盐清除活性。柱层析循环联合法可以同时提取多糖、总黄酮、总皂苷,且提取效率较高,为刺玫果产品的开发提供依据。