Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of nine corticosteroid residues in bovine liver samples

A liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography-negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. All the corticosteroids can be detected at a concentration around 1 μg kg(-1); the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.

Synthesis, binding and cellular uptake properties of oligodeoxynucleotides containing cationic bicyclo-thymidine residues

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6′)-β position is described. Compared to unmodified DNA these oligonucleotides show Tm-neutral behavior when paired against complementary DNA and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic distribution which was not observed in the case of an unmodified control oligonucleotide.

Identification of key residues in virulent canine distemper virus hemagglutinin that control CD150/SLAM-binding activity

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by beta-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms beta2 and theta

AIMS/HYPOTHESIS: Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor beta-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity. METHODS: Tyrosine kinase assay and transfection studies. RESULTS: In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms beta2 and theta. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C. CONCLUSION/INTERPRETATION: The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms beta2 and theta on insulin receptor autophosphorylation.

Insoluble Residues of the Lower Mississippian Limestones of the Madison Group

The correlation of non-fossiliferous drill samples is one of the difficult problems that is en­countered in sub-surface stratigraphy. In order to truly correlate a formation, it must have some dis tinctive features and have an areal persistence of these features. These requirements are probably met best by limestone.

Insoluble Residues of Five Sections of the Amsden Formation

The term, "insoluble residue," as used in this report is that portion of the original rock sample remaining after the sample has been digested by a mixture of one part hydrocloric acid and two parts water. The remains or insoluble residue from this acid treatment may vary from nothing to I00 percent.

Histological assessment of testicular residues in lambs and calves after Burdizzo castration

To assess the reliability of the Burdizzo procedure for castrating calves and lambs, testicular tissue from 63 bull calves (15 intact and 48 castrated) and 69 male lambs (35 intact and 34 castrated) was collected at slaughter and assessed histologically. The bull calves were castrated at either one, four to five or 12 to 16 weeks of age and the lambs at either one or 10 weeks. There was clear evidence of spermatogenesis in testicular tissue from all the intact animals. In the samples from the calves that had been castrated at 12 to 16 weeks functional testicular tissue was completely lacking. However, there was evidence of spermatogenesis and steroidogenesis in the calves that had been castrated at one week or four to five weeks, respectively. Failure to achieve complete involution of the testicular parenchyma was observed in the majority of lambs, irrespective of the age at which they had been castrated.

Nutritive Value of the Crop Residues from bt-Corn Hybrids and Their Effects on Performance of Grazing Beef Cows

One non bt-corn hybrid (Pioneer 3489) and three btcorn hyrids (Pioneer 34RO7, Novartis NX6236, and Novartis N64-Z4) were planted in replicated 7.1-acre fields. After grain harvest, fields were stocked with 3 mature cows in midgestation to be strip-grazed as four paddocks over 126 days. Six similar cows were allotted to replicated drylots. All cows were fed hay as necessary to maintain a condition score of 5 on a 9-point scale. Cows were condition-scored biweekly and weighed monthly. Forage yield and weathering losses were determined by sampling one 4-m2 location per grazed or ungrazed paddock in each field with a minimum total of 2 locations of grazed or ungrazed forage per field. To measure forage selection during grazing, samples of grazed forage were collected from the rumen of one fistulated steer that grazed for 2 hours after ruminal evacuation. Non-bt-corn hybrids had greater (P<.05) infestation of corn borers in the upper stalk, lower stalk and ear shank than bt-corn hybrids. However, there were no differences in grain yields or dropped grain between hybrids. Crop residue dry matter, organic matter and in vitro digestible dry matter yields at the initiation of grazing did not differ between corn hybrids. Dry matter, organic matter and in vitro digestible dry matter losses tended (P<.10) to be greater from the NX6236 and N64-Z4 hybrids than from the 3489 and 34RO7 hybrids and were greater (P<.05) from grazed than non-grazed areas of the fields. At the initiation of grazing, dry matter concentrations of the crop residues from the NX6236 and N64-Z4 hybrids tended to be lower than those from the 3489 and 34RO7 hybrids. Crop residues from the NX6236 and N64-74 hybrids had lower concentrations of acid detergent fiber (P<.05) and acid detergent lignin (P=.07) and higher concentrations of in vitro digestible organic matter than the 3489 and 34RO7 hybrids. Over the grazing season, corn hybrid did not affect mean rates of change in forage composition. The concentration of in vitro digestible organic matter in forage selected by steers after two weeks of grazing did not differ. However, steers grazing corn crop residues consumed forage with higher (P<.05) concentrations of neutral detergent fiber, acid detergent fiber, and acid detergent insoluble nitrogen than steers fed hay. The acid detergent fiber concentration of forage selected by steers grazing the 3489 and N64-Z4 hybrids was lower (P < .05) than concentrations from the 34RO7 and NX6236 hybrids. In order to maintain similar body condition score changes, cows grazing crop residues from the 3489, 34RO7, NX6236, and N64-Z4 hybrids required 650, 628, 625, and 541 kg hay DM/cow compared with a hay requirement of 1447 kg hay DM/cow for cows maintained in a drylot.

Use of Stockpiled Berseem Clover as a Supplement for Grazed Corn Crop Residues

In the fall of 1994, mature Charolais cross cows in midgestation were allotted to duplicate 15 acre fields containing corn crop residues or a 2-to-1 mixture of corn crop residues and berseem clover planted in 3 strips at an allowance of 2.5 acres/cow for a 140 day wintering season. Similar cows were allotted duplicate drylots. All cows were fed hay as necessary to maintain a body condition score of 5. Cows grazing corn crop residues with or without berseem clover required 2596 pounds less hay per cow than cows maintained in a drylot. There was no difference in the amounts of hay required by cows grazing corn crop residues alone or with berseem clover. Initial organic matter yield of berseem clover was nearly that of corn crop residues and did not decrease as rapidly as corn crop residues. Berseem clover had a higher organic matter digestibility than corn crop residues at the initiation of grazing. Organic matter digestibility of berseem clover, however, decreased more rapidly than corn crop residues because of weathering during the winter.

Efficiency of risk-based vs . random sampling for the monitoring of tetracycline residues in slaughtered calves in Switzerland

In all European Union countries, chemical residues are required to be routinely monitored in meat. Good farming and veterinary practice can prevent the contamination of meat with pharmaceutical substances, resulting in a low detection of drug residues through random sampling. An alternative approach is to target-monitor farms suspected of treating their animals with antimicrobials. The objective of this project was to assess, using a stochastic model, the efficiency of these two sampling strategies. The model integrated data on Swiss livestock as well as expert opinion and results from studies conducted in Switzerland. Risk-based sampling showed an increase in detection efficiency of up to 100% depending on the prevalence of contaminated herds. Sensitivity analysis of this model showed the importance of the accuracy of prior assumptions for conducting risk-based sampling. The resources gained by changing from random to risk-based sampling should be transferred to improving the quality of prior information.