Changes in Acute-Phase Proteins in Plasma during the Periparturient Period of Dairy Goats.

The present study was conducted regarding four acute-phase proteins (APPs) including C-reactive protein (CRP), ceruloplasmin (CP), serum amyloid A (SAA), and haptoglobin (HP) in dairy goats during the periparturient period. The aim of this study was to detect the changes in APPs in plasma during the periparturient period of healthy dairy goats. Guanzhong dairy goats with no other symptoms (n = 15) were selected on the basis of their blood calcium (Ca) and ß-hydroxybutyrate (BHBA) concentration. The plasma was collected once a week for ±3 weeks delivery. The concentrations of the four APPs mentioned above were determined using goat-specific ELISA kits. The results showed the CRP level in plasma decreased from 3 weeks to 1 week antepartum and increased later until 1 week postpartum and then decreased to a similar level with antepartum between 1 and 3 weeks postpartum. The content of CP showed a decline in 3 weeks before parturition and an upward trend between 1 week antepartum and 3 weeks postpartum. The SAA concentration decreased from 3 weeks antepartum to 2 weeks postpartum and rebounded later. The level of HP decreased during 3 weeks before parturition and increased until 1 week postpartum, then reached a stable value. Clear variation range and rules of APPs contribute to perinatal health monitoring of dairy goats.

NEFA Promotes Autophagosome Formation through Modulating PERK Signaling Pathway in Bovine Hepatocytes.

During the perinatal period, the abnormally high plasma non-esterified fatty acids (NEFA) concentration caused by the negative energy balance (NEB) can impose a significant metabolic stress on the liver of dairy cows. Endoplasmic reticulum (ER) stress is an important adaptive response that can serve to maintain cell homeostasis in the event of stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is the most rapidly activated cascade when ER stress occurs in cells and has an important impact on the regulation of hepatic lipid metabolism and autophagy modulation. However, it is unknown whether NEFA can affect autophagy through modulating the PERK pathway, under NEB conditions. In this study, we provide evidence that NEFA treatment markedly increased lipid accumulation, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the expression of glucose-regulated protein 78 (Grp78), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). More importantly, NEFA treatment can cause a substantial increase in the protein levels of autophagy-related gene 7 (ATG7), Beclin-1 (BECN1), sequestosome-1 (p62), and microtubule-associated protein 1 light chain 3 (LC3)-II, and in the number of autophagosomes in primary bovine hepatocytes. The addition of GSK2656157 (PERK phosphorylation inhibitor) can significantly inhibit the effect of NEFA on autophagy and can further increase lipid accumulation. Overall, our results indicate that NEFA could promote autophagy via the PERK pathway in bovine hepatocytes. These findings provide novel evidence about the potential role of the PERK signaling pathway in maintaining bovine hepatocyte homeostasis.

Surrogate Indexes of Insulin Resistance in Dairy Goats: Transitional Variation in Subclinical Hyperketonemia.

BACKGROUND: Dairy goats are highly susceptible to subclinical hyperketonemia (SCHK) during the transition period. This study aimed to compare the variation in metabolic parameters and surrogate indexes of insulin resistance (sIR) between goats with SCHK and clinically healthy (HEAL) goats during the transition period. METHODS: Twenty Guanzhong dairy goats were assorted to HEAL (n = 10) and SCHK (n = 10) groups according to the blood ß-hydroxybutyrate (BHBA) concentrations. The blood samples were taken from the jugular vein of each goat at -3, -2, -1, 0 (partum), +1, +2, and +3 weeks relative to kidding to analyses GLU and INS. The sIR was calculated from blood metabolic parameters. RESULTS: Compared with the HEAL goats, the insulin concentrations were significantly higher in SCHK goats during the first three weeks postpartum. The QUICKI, revised QUICKI (RQUICKI), and RQUICKIBHBA were significantly lower in goats with SCHK at 1 week postpartum, while the homeostasis model assessment-IR (HOMA-IR) was significantly higher. CONCLUSION: Goats with SCHK made more efforts through elevated insulin levels at early lactation than HEAL goats, thereby maintaining the normal glucose concentrations.

Oxidative status in dairy goats: periparturient variation and changes in subclinical hyperketonemia and hypocalcemia.

BACKGROUND: A better comprehension of the redox status during the periparturient period may facilitate the development of management and nutritional solutions to prevent subclinical hyperketonemia (SCHK) and subclinical hypocalcemia (SCHC) in dairy goats. We aimed to evaluate the variation in the redox status of dairy goats with SCHK and SCHC during their periparturient periods. Guanzhong dairy goats (n = 30) were assigned to SCHK (n = 10), SCHC (n = 10), and healthy (HEAL, n = 10) groups based on their blood ß-hydroxybutyrate (BHBA) and calcium (Ca) concentrations. Blood were withdrawn from goats every week from 3 weeks before the expected parturition date to 3 weeks post-kidding. On the same day, the body condition scores (BCS) were evaluated, and the milk yield was recorded for each goat. The metabolic profile parameters and the indicators of oxidative status were determined by using the standard biochemical techniques. RESULTS: In comparison with the HEAL goats, SCHK and SCHC goats presented with a more dramatic decline of BCS post-kidding and a significant decrease in the milk yield at 2- and 3-weeks postpartum, ignoring the obvious increase at 1-week postpartum. The levels of non-esterified fatty acids (NEFA) peaked at parturition, exhibiting significantly higher levels from 1-week prepartum to the parturition day in the SCHK and SCHC groups. The malondialdehyde (MDA) concentration was increased in the SCHK goats from 1-week antepartum until 3-weeks postpartum, with its concentration being significantly higher in the SCHC goats at parturition. The hydrogen peroxide (H2O2) concentration was significantly lower in the SCHK and SCHC goats from 2-weeks antepartum to 1-week post-kidding. The total antioxidant capacity (T-AOC) and the superoxide dismutase (SOD) level were decreased at 1-week antepartum in the SCHK and SCHC goats, respectively. The glutathione peroxidase (GSH-Px) level was increased in the SCHK and SCHC goats during the early lactation period. CONCLUSIONS: The SCHK and SCHC goats exerted more efforts to maintain their redox homeostasis and to ensure the production performance than the HEAL goats during their periparturient period, probably owing to more intense fat mobilization and lipid peroxidation in the former.

Elucidation of the mechanism of NEFA-induced PERK-eIF2α signaling pathway regulation of lipid metabolism in bovine hepatocytes.

During the periparturient transition period, negative energy balance (NEB) characterized by high concentrations of non-esterified fatty acids (NEFA) may cause fatty liver and ketosis in dairy cows. Previous studies have shown that the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the endoplasmic reticulum stress (ERS) response plays an important role in lipid metabolism in hepatocytes. This study, therefore, investigated the role of the PERK-branch in NEFA-induced fatty liver. Different concentrations of NEFA or GSK2656157 (a novel catalytic inhibitor of PERK) were used to treat hepatocytes isolated from calves. The NEFA treatment significantly increased the triacylglycerol (TG) content, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the abundance of glucose-regulated protein 78 (Grp78), C/EBP homologous protein (CHOP), sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), apolipoprotein B (APOB), and the low-density lipoprotein receptor (LDLR). Compared with the 1.2 mM NEFA group, inhibition of PERK activity further increased the TG content in hepatocytes, the very-low-density lipoprotein (VLDL) content in the supernatant and the protein abundance of APOB while reducing the expression and nuclear levels of SREBP-1c and PPARα, as well as the expression of CPT1A and CPT2. In conclusion, the results showed that the NEFA-induced PERK-eIF2α signaling pathway promotes lipid synthesis, lipid oxidation, but inhibits the assembly and secretion of VLDL. Therefore, during the transition period, the activation of the PERK-eIF2α signaling pathway in the liver of dairy cows could defeat the acid-induced lipotoxicity and provide energy to alleviate NEB.

Nitrogen Supply Affects Yield and Grain Filling of Maize by Regulating Starch Metabolizing Enzyme Activities and Endogenous Hormone Contents.

This study aimed to examine the effect of nitrogen (N) application rate and time on yield, grain filling, starch metabolizing enzymes, and hormones of maize based on a long-term field experiment initiated in 2012. The total N fertilizer dose [(0 (N0), 100 (N1), 200 (N2), and 300 (N3) kg N ha-1] was split into two (T1, one-third at sowing and two-thirds at the six-leaf stage) or three (T2, one-third each at sowing, six-leaf, and eleven-leaf stage) times application. The results showed that the highest yield was obtained under N3T2, N2T1, and N3T2 in 2018, 2019, and 2020, which was 222.49, 185.31, and 194.00% than that of N0 in each year, respectively. N2 and N3 significantly increased the yield through enhancing ears ha-1, grains per plant, and 100-grain weight; however, N2 and N3 did not show a significant difference in yield and above-yield components. In addition, N application time did not significantly change yield under the same N rate. N0 limited the activities of starch metabolizing enzymes, resulting in insufficient accumulation of sucrose and starch. The contents of indole-3-acetic acid, cytokinin, abscisic acid, and gibberellin were decreased under N0 during grain filling. The average grain-filling rate and maximum grain-filling rate (G max) and grain weight increment achieving G max increased under N2 and N3, and the grain-filling parameters were positively correlated with 100-grain weight. In conclusion, 200 kg N ha-1 with one-third application at sowing and two-thirds application at the six-leaf stage is a suitable N supply way to improve starch metabolizing enzymes, regulate hormone content, and enhance grain-filling rates, and thus increasing the maize yield in the semiarid Loess Plateau of China.

Angiopoietin-like protein 4 promotes very-low-density lipoprotein assembly and secretion in bovine hepatocytes in vitro.

In dairy cows, fatty liver is one of the most common metabolic diseases that occurs during the periparturient period. Angiopoietin-like protein 4 (ANGPTL4) is a well-known downstream target of peroxisome proliferator-activated receptors (PPARs), which regulate the glucose and fatty acid metabolisms. The inhibition of lipoprotein lipase (LPL) activity interferes with the storage of triglycerides (TG) in adipocytes, which plays an essential role in lipid metabolism in rodents. However, it remains unclear whether ANGPTL4 is involved in the pathological process of fatty liver in dairy cows as a result of the regulation of the hepatocellular lipid transport system. This study intended to investigate the effect of ANGPTL4 on the very-low-density lipoprotein (VLDL) assembly and secretion in bovine hepatocytes. Bovine hepatocytes were isolated using a modified two-step perfusion and collagenase digestion process, and treated with different concentrations of ANGPTL4 (0, 4, 12, and 24 ng/ml) for 24 hr. The results showed that a high concentration of ANGPTL4 could significantly increase the extracellular concentration of VLDL while reducing the intracellular content of TG. Thus, it was confirmed that ANGPTL4 could promote the transport of TG in the form of VLDL by partially regulating the expression of related proteins in hepatocytes, thereby contributing to the partial adaptive regulation of lipid transport in dairy cows.

The role of the 4''-hydroxyl on motilin agonist potency in the 9-dihydroerythromycin series.

The role of the erythromycin 4''-hydroxyl group has been explored on the motilin agonist potential in the 9-dihydroerythromycin series of motilides. The compounds show potencies 2- to 4-fold superior to the corresponding hydroxylated compounds. The relationship is maintained when the 9-hydroxyl is alkylated to generate the corresponding 4''-deoxy-9-O-acetamido-9-dihydroerythromycins. However, concomitant with this increase in potency is an increase in hERG inhibition.

9-Dihydroerythromycin ethers as motilin agonists--developing structure-activity relationships for potency and safety.

A series of derivatives of the amine of 9-dihydro-9-O-ethylamino-N-desmethyl-N-isopropyl erythromycin A derivatives were synthesized as motilin agonists. The compounds were developed for potency without showing antibacterial activity and inhibition of the hERG potassium channel. The formamide of the amide series was found to show the optimal combination of properties relative to carbamates, ureas, thioureas, and amines. This prompted an investigation of heterocyclic isosteres for the amide. In this series the triazole had the optimal combination of properties. From the study, two compounds met the criteria for detailed pharmacokinetic studies.

9-Dihydroerythromycins as non-antibiotic motilin receptor agonists.

A series of 9-dihydroerythromycin A and B analogues with modification of the desosamine nitrogen have been synthesized and screened for motilin agonist activity, antibiotic activity, tachyphylaxis and hERG channel current inhibition. Small alkyl groups resulted in the potency while compounds with a primary or secondary amine resulted in the low motilin agonist potency. Several compounds were identified as non-antibiotic motilin receptor agonists with minimal tachyphylaxis and low hERG interaction.

Structure-activity relationships of 9-substituted-9-dihydroerythromycin-based motilin agonists: optimizing for potency and safety.

A series of 9-dihydro-9-acetamido-N-desmethyl-N-isopropyl erythromycin A analogues and related derivatives was generated as motilin agonists. The compounds were optimized for potency while showing both minimal antibacterial activity and hERG inhibition. As the substituent on the amide was increased in lipophilicity the potency and hERG inhibition increased, while polar groups lowered potency, without significantly impacting hERG inhibition. The N-methyl acetamide 7a showed the optimal in vitro profile and was probed further by varying the chain length to the macrocycle as well as changing the macrocycle scaffold. 7a remained the compound with the best in vitro properties.

Potent cytotoxic C-11 modified geldanamycin analogues.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the activity of Hsp90, an important target for treatment of cancers. In an effort to identify analogues of geldanamycin (GDM) with properties superior to those of 17-AAG, we synthesized C-11 modified derivatives of GDM including ethers, esters, carbazates, ketones, and oximes and measured their affinity for Hsp90 and their ability to inhibit growth of human cancer cells. In accordance with crystal structures reported for complexes of GDMs with Hsp90, bulky groups attached to C-11 interfered with Hsp90 binding while smaller groups such as 11-O-methyl allowed Hsp90 binding. In addition, these analogues also showed in vitro cytotoxicity against human cancer cell lines. Esterification of the 11-OH of 17-AAG eliminated Hsp90 binding in vitro. The readily hydrolyzed esters acted as prodrugs during the measurement of cytotoxicity. Thus, during these experiments, the esters were hydrolyzed, releasing 17-AAG. Several 11-O-methyl-17-alkylaminogeldanamycin analogues were identified with improved potency relative to 17-AAG.

The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261.

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

Heterologous production of epothilone C and D in Escherichia coli.

The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster,epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.

Alteration of insulin-like growth factor-1 expression after middle cerebral artery occlusion in monkeys and rats: complementary DNA microarray, immunohistochemistry, and in situ hybridization studies.

It is well established that insulin-like growth factor (IGF)-1 has potent neuroprotective effects on cerebral ischemia in the rat and sheep model. In order to investigate whether it has neuroprotective effects on brain insult in human stroke, as one part of serial subhuman primate stroke research, the present study was designed to observe whether IGF-1 messenger RNA (mRNA) and protein is expressed in middle cerebral artery occlusion in monkeys and rats. A total of 12,800 dots complementary DNA microarray, in situ hybridization, and immunohistochemistry were used. Complementary DNA microarray showed that among the nearly 8000 genes, approximately 8% of the total number of genes examined was affected after ischemia/reperfusion injury especially in the growth factor family including IGF-1 in the ischemic region. The decreased IGF-1 mRNA and protein expression was found in the insular striatum, but there was an increased mRNA expression and unchanged protein expression in the hippocampus 24 hours after ischemia. The results suggested that IGF-1 might contribute to the neuroprotective pathway in a pattern different from that of rats, and it might play a role in protection of ischemic injured neuronal cells after monkey focal cerebral ischemia.

A reversible middle cerebral artery occlusion model using intraluminal balloon technique in monkeys.

BACKGROUND: This study was designed to develop a simple, reversible primate middle cerebral artery (MCA) occlusion (MCAO) model. METHODS: MCAO was achieved by inserting a specially designed intraluminal balloon catheter from the external carotid artery, then to the internal carotid artery, and finally into the proximal segment of MCA using 13 adolescent monkeys (Macaca mulatta) weighing 4.25 +/- 0.23 kg. The infarction was confirmed by magnetic resonance imaging at 3, 7, and 23 hours after MCAO. RESULTS: Local cerebral blood flow in the caudate nucleus decreased remarkably. Post-MCAO electroencephalographic monitoring showed the inhibition of electroencephalography with slow waves distinctly increasing. Neurologic evaluation demonstrated persistent deficits in the ischemic group. Neuropathologic examinations were consistent with the neurologic evaluations. CONCLUSION: The results suggested that this reversible monkey model resembled a human stroke and should offer a reasonable alternative to craniotomy and the extrinsic ligation of the MCA in the laboratory investigation of focal reversible cerebral ischemia.

Structure-based design of 7-carbamate analogs of geldanamycin.

The 7-carbamate groups of geldanamycin and its 17-(2-dimethylaminoethyl)amino-17-demethoxy derivative (17-DMAG) bind the N-terminal domain of Hsp90 by establishing a network of hydrogen bonds which involve four buried water molecules. In this study, a structure-based approach was used to investigate the effects of displacing some of these waters by modification of the 7-carbamate. A general loss of binding to human Hsp90 was observed, except for replacement of the carbamate with a hydroxamate group which gave an analog with weak activity. Modeling of Hsp90-ligand interactions suggested that the hydroxamate was not able to displace the buried water molecules, while bulkier substituents able to do so proved inactive.

Desensitization of the human motilin receptor by motilides.

Tachyphylaxis may have contributed to the failure of the motilide ABT-229 [N-ethyl, N-methyl 4'' deoxy erythromycin (EM)-B enolether] in clinical trials. We compared the desensitizing potency of structurally related motilides [EM-A, EM-A enolether (ME4), N-ethyl, N-methyl EM-A (ME36), EM-B enolether (ME67), N-ethyl, N-methyl EM-A enolether (EM523), ABT-229 and 4'' deoxy EM-A enolether (KOS1326)] in a Chinese hamster ovary (CHO)-K1 cell line expressing the human motilin receptor (MTLR) and in rabbit duodenal segments. CHO-MTLR cells were preincubated with motilides prior to stimulation with motilin. The negative logarithm of the preincubation concentration reducing the maximal motilin-induced Ca(2+) flux to 50% was calculated (pDC(50)). Internalization was visualized in CHO-K1 cells containing an enhanced green fluorescent protein (EGFP)-tagged MTLR and quantified in binding experiments. The contractile response of repeated stimulations was measured in duodenal segments. In CHO-MTLR cells, the pDC(50) was ABT-229 (8.78) > motilin (7.77) > EM-A (4.78), different from their order of potency to induce Ca(2+) release (pEC(50)): motilin (9.39) > ABT-229 (8.46) > EM-A (7.11). In cells with the EGFP-tagged MTLR, ABT-229 decreased membrane fluorescence by 25 +/- 2% compared with 16 +/- 2% for motilin and 8 +/- 2% for EM-A. Binding studies confirmed that EM-A did not induce MTLR internalization (residual binding 96 +/- 4% compared with motilin, 31 +/- 3% and ABT-229, 21 +/- 1%). Comparison of the pDC(50) and pEC(50) values of the other motilides ME4 (5.90; 8.08), ME67 (6.03; 8.12), ME36 (3.32; 6.62), EM-523 (6.02; 8.22), and KOS1326 (7.32; 8.14) suggested that the strong desensitizing properties of ABT-229 are mostly related to the removal of the 4''-OH of the cladinose sugar. The decline of the contractile response in duodenal segments correlated with the pDC(50). The ability to desensitize and internalize the MTLR is not only determined by potency. This may be an important criterion for the development of a clinically useful compound.

Isolation and characterization of novel geldanamycin analogues.

New geldanamycin analogues with novel structures arising from direct microbial bioconversion and a genetically engineered geldanamycin producer were isolated and characterized. Three compounds, 15-hydroxygeldanamycin, a tricyclic geldanamycin analog (KOSN-1633), and methyl-geldanamycinate), were isolated after geldanamycin was added to a growing culture of the herbimycin producing strain-Streptomyces hygroscopicus AM-3672. Two related compounds, 17-formyl-17-demethoxy-18-O,-21-O-dihydrogeldanamycin and 17-hydroxymethyl-17-demethoxygeldanamycin were isolated from S. hygroscopicus NRRL 3602/pKOS279-78, a geldanamycin-producing strain containing various genes isolated from S. hygroscopicus AM-3672. Compared with geldanamycin, these five new compounds exhibited reduced cytotoxicity against SKBr3 cancer cells.

Synthesis and biological activities of novel 17-aminogeldanamycin derivatives.

Geldanamycin interferes with the action of heat shock protein 90 (Hsp90) by binding to the N-terminal ATP binding site and inhibiting an essential ATPase activity. In a program directed toward finding potent, water soluble inhibitors of Hsp90, we prepared a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogs, and compared their affinity for Hsp90, ability to inhibit growth of SKBr3 mammalian cells, and in selected cases, water solubility. Over 20 analogs showed cell growth inhibition potencies similar to that of 17-allylamino-17-demethoxygeldanamycin (17-AAG), the front-runner geldanamycin analog that is currently in multiple clinical trials. Many of these analogs showed water solubility properties that were desirable for formulation. One of the most potent and water-soluble analogs in the series was 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG), which was independently prepared by the NCI and will soon enter clinical trials. Importantly, the binding affinity of these analogs to the molecular target Hsp90 does not correlate well with their cytotoxicity in SKBr3 cells.