Kerry G. Bemis

Label-free mass spectrometry-based protein quantification technologies in proteomic analysis

Major technological advances have made proteomics an extremely active field for biomarker discovery and validation in recent years. These improvements have lead to an increased emphasis on larger scale, faster and more efficient methods for protein biomarker discoveries in human tissues, cells and biofluids. However, most current proteomic methodologies for biomarker discovery and validation are not highly automated and generally labour intensive and expensive. Improved automation as well as software programs capable of handling a large amount of data are essential in order to reduce the cost of discovery and increase the throughput. In this review, we will discuss and describe the label-free mass spectrometry-based protein quantification technologies and a case study utilizing one of these methods for biomarker discovery.

Experimental Designs for Selecting Molecules from Large Chemical Databases

Recent developments in high-throughput screening and combinatorial chemistry have generated interest in experimental design methods to select subsets of molecules from large chemical databases. In this manuscript three methods for selecting molecules from large databases are described:  edge designs, spread designs, and coverage designs. Two algorithms with linear time complexity that approximate spread and coverage designs are described. These algorithms can be threaded for multiprocessor systems, are compatible with any definition of molecular distance, and may be applied to very large chemical databases. For example, ten thousand molecules were selected using the maximum dissimilarity approximation to a spread design from a sixty-dimensional simulated molecular database of one million molecules in approximately 6 h on a UNIX workstation.

Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphines affinity for opioid receptors labeled in vitro with 3H-naloxone or 3H-D-Ala2-D-Leu5-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties.

Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand, DAla2DLeu5 enkephalin in mouse brain

The binding characteristics of the delta opioid receptor ligand, 3H-D-Ala2-D-Leu5 enkephalin, were markedly altered in brains obtained from mice fed an ethanol-containing diet for five days. Control mice exhibited both a high and low affinity site for 3H-D-Ala2-D-Leu5 enkephalin, whereas those consuming the ethanol diet were found to possess only one binding site. This singular site has an intermediate KD value with an increase in receptor number when compared to the high and low affinity sites observed in control mice. The in vitro addition of ethanol to a brain membrane preparation obtained from untreated mice, at a concentration equivalent to that found in the blood of the ethanol-treated mice, did not markedly affect D-Ala2-D-Leu5 enkephalin binding characteristics. No alteration in the binding characteristics of 3H-naloxone, a mu receptor ligand, was noted following five days of ethanol consumption. Mice maintained on the ethanol-containing diet were tolerant to the activity-stimulating effects of acute ethanol administration. These results suggest that mice consuming an ethanol diet in sufficient quantities to render them tolerant exhibit a specific loss of a 3H-D-Ala2-D-Leu5 enkephalin binding site, while the binding of 3H-naloxone was unchanged.

Elevation of Serum Corticosterone in Rats by Dopamine Agonists Related in Structure to Pergolide

Two chemical analogs of pergolide were examined to test further the idea that pergolide elevates serum corticosterone concentration in rats by activation of brain dopaminergic receptors. LY116467, which contains an N-methyl substituent in place of the N-n-propyl substituent in pergolide, was less potent than pergolide in lowering brain levels of 3,4-dihydroxyphenylacetic acid (Dopac), the metabolite of dopamine. LY116467 increased serum corticosterone concentration in rats at a dose of 3 mg/kg (a higher dose than is required for pergolide), and the effect was prevented by spiperone pretreatment. LY141865, which has been reported to differ from pergolide in not activating dopamine-sensitive adenylate cyclase and which was found in this study to have much less affinity for serotonin receptors than does pergolide, increased serum corticosterone in much the same manner as pergolide, only slightly higher doses being required. The effect of LY141865 was prevented by pretreatment with haloperidol but not domperidone. Both haloperidol and domperidone increased serum prolactin concentration when given alone or in combination with LY141865, indicating they were both capable of blocking peripheral (pituitary) dopamine receptors. In contrast, haloperidol but not domperidone caused a marked elevation in brain levels of Dopac and of homovanillic acid and prevented the lowering of these brain dopamine metabolites by LY141865. The ability of LY141865 to increase serum corticosterone concentration was attenuated in rats that had received four daily injections of pergolide mesylate, indicating corss-tolerance had occurred. These results strengthen the hypothesis that activation of brain dopaminergic receptors leads to increased serum corticosterone concentration in rats.

Porcine pancreatic phospholipase A2-induced contractions of guinea pig lung pleural strips

The contractile nature of porcine pancreatic phospholipase A2 (PLA2) was characterized on paired pleural strips obtained from guinea pig lung. PLA2 (0.003-10 U/ml) produced concentration-related contractile responses which were sensitive to various drugs. The major component of the PLA2-induced contractions was derived from products of the cyclooxygenase pathway since a cyclooxygenase inhibitor or the combination of a thromboxane synthetase inhibitor and a thromboxane receptor antagonist produced a 54-65% reduction of the contractile responses. 5-Lipoxygenase products contributed to a smaller component of the PLA2-induced responses since 5-lipoxygenase inhibitors or the combination of a leukotriene (LT) B4 receptor antagonist and an LTD4/LTE4 receptor antagonist only suppressed the maximal responses 22-32%. PLA2-induced contractile responses were nearly abolished by altering both sides of the arachidonic acid cascade simultaneously. In contrast, a PAF receptor antagonist, a histamine (H1) receptor antagonist and an acetylcholine receptor antagonist, failed to significantly reduce PLA2-induced responses. These results demonstrate that exogenous administration of porcine pancreatic PLA2 produced concentration-dependent contractions of pleural strips mediated through the generation of eicosanoids.

Antihypertensive effects of fluoxetine and L-5-hydroxytryptophan in rats.

Abstract The combination of fluoxetine (10 mg/kg) and L-5-hydroxytryptophan (5-HTP) (10 mg/kg) significantly lowered blood pressure in spontaneously hypertensive rats and in rats made hypertensive by treatment with deoxycorticosterone (DOCA) and saline. Fluoxetine alone also had a significant effect on blood pressure in DOCA hypertensive rats, but not as great an effect as the combination. Since fluoxetine is an inhibitor of serotonin reuptake and 5-HTP is the serotonin precursor, the antihypertensive effect of this drug combination strengthens previous evidence that serotonin neurons have a role in the central regulation of blood pressure.

Assessment of working memory in rats using spatial alternation behavior with variable retention intervals: effects of fixed-ratio size and scopolamine

The effects of fixed-ratio (FR) size, scopolamine, and the interactions between FR size and scopolamine were investigated in male F344 rats on working memory as assessed by spatial alternation behavior maintained under FR schedules of food presentation where the interval between trials was varied among values of 2, 4, 8, 16, and 32 s within each session. The magnitude of the FR size on the correct and incorrect levers was varied systematically from 1 response to 2, 4, 8, or 16 responses in order to determine whether the FR size influenced either the percentage of correct responding, rates of responding, or both. Under the primary baseline condition, that is when the FR size on both the correct and incorrect levers was one response (designated FR1 FR1), the percentage of correct responses decreased with increasing retention interval duration. Increasing the FR size on the correct lever produced FR-dependent increases in the percentage of correct responding as well as in rates of responding. Increasing the FR size on the incorrect lever produced FR-dependent decreases in correct responding, but had little effect on rates of responding. Dose-effect curves for scopolamine were determined on performance maintained under FR values on the correct and incorrect levers, respectively, of FR1 FR1, FR1 FR10, FR10 FR1, and FR10 FR10. In general, scopolamine produced dose-related decreases in the percentage of correct responding, although the magnitude of the effects of scopolamine varied not only with dose, but also with the length of the retention interval and with changes in FR size. Rates of responding during trials were dose-dependently decreased by scopolamine under all schedule parameters. The present results are consistent with the interpretation that scopolamine can selectively impair time-dependent memory processes such as working memory, but also can impair time-independent variables which affect performance, dependent on dose and schedule maintaining the behavior.

Analysis of the beta1 and beta2 adrenoceptor interactions of the partial agonist, clenbuterol (NAB365), in the rat jugular vein and atria

SummaryThe potent bronchodilator, clenbuterol, was compared to other beta adrenoceptor agonists with regard to affinity and efficacy for interaction with beta1 and beta2 adrenoceptors in the rat jugular vein and atria. Clenbuterol was a potent partial beta adrenoceptor agonist in both tissues based on the following observations: 1. Maximal relaxation of the jugular vein and increases in atrial rate to clenbuterol were less than maximal responses to other beta adrenoceptor agonists. 2. Clenbuterol antagonized responses to the stronger agonist, isoproterenol, in both tissues and 3. the equilibrium dissociation constant for clenbuterol approximated the ED50 concentration for vascular relaxation and increase in atrial rate, a characteristic of some, but not all, partial agonists. Relative to other beta adrenoceptor agonists, clenbuterol showed high affinity toward both beta1 and beta2 adrenoceptors and selectivity toward beta2 adrenoceptors. Equilibrium dissociation constants were 38 and 6.3 nM for beta1 and beta2 adrenoceptors, respectively. The high affinity of clenbuterol toward beta1 and beta2 adrenoceptors was coupled to a low relative efficacy of clenbuterol to activate either beta1 or beta2 adrenoceptors. Most beta2 adrenoceptor agonists such as isoproterenol or salbutamol require approximately 1–3% adrenoceptor occupation for 40–50% relaxation of the jugular vein whereas clenbuterol required approximately 100% adrenoceptor occupation for a similar response. Thus, based on our analysis, the high agonist potency of clenbuterol results primarily from the high affinity toward beta adrenoceptors rather than efficient activation of the adrenoceptor as occurs with isoproterenol or salbutamol.

Inhibitory Effect of Warfarin on the Metastasis of the PAIII Prostatic Adenocarcinoma in the Rat

The PAIII rodent metastatic prostatic adenocarcinoma model was employed to evaluate the effects of dietary warfarin, a prototypic antagonist of thrombin generation on the lymphatic and pulmonary metastases of the tumor from the tail site of subcutaneous transplantation in male Lobund Wistar (LW) rats. In addition, the anticoagulant effects of warfarin were determined in the same animals. Warfarin, administered in the diet at concentrations equivalent to 0.063, 0.125 or 0.250 mg./kg. b.w. for 30 days had no effect on final body weight, gluteal or iliac lymph node weights. Significant (p less than 0.05) dose-dependent extensions of whole blood prothrombin (WBPT), activated partial thromboplastin (WBAPTT) and clotting times (WBCT) over control values were observed with warfarin treatment. Preliminary studies demonstrated that the 0.500 mg./kg. dose produced 50 per cent mortality at +14 days. Warfarin produced significant (p less than 0.05) dose-dependent decreases in the number of PAIII pulmonary metastases as indicated by reductions in dry lung weights and lung colony numbers when compared to untreated tumor-bearing controls. While the therapeutic index of warfarin is a limiting factor in clinical use as an antimetastatic agent, these results suggest that compounds capable of altering hemostatic mechanisms may be potential inhibitors of tumor metastasis. The PAIII prostatic adenocarcinoma model may be a useful system to quantitatively evaluate potential antimetastatic and cytotoxic agents.