Development of a fluorescent ligand-binding assay using the AcroWell filter plate

One of the most powerful tools for receptor research and drug discovery is the use of receptor-ligand affinity screening of combinatorial libraries. Early work involved the use of radioactive ligands to identify a binding event; however, there are numerous limitations involved in the use of radioactivity for high throughput screening. These limitations have led to the creation of highly sensitive, nonradioactive alternatives to investigate receptor-ligand interactions. Pall Gelman Laboratory has introduced the AcroWell, a patented low-fluorescent-background membrane and sealing process together with a filter plate design that is compatible with robotic systems. Taken together, these allow the AcroWell 96-well filter plate to detect trace quantities of lanthanide-labeled ligands for cell-, bead-, or membrane-based assays using time-resolved fluorescence. Using europium-labeled galanin, we have demonstrated that saturation binding experiments can be performed with low-background fluorescence and signal-to-noise ratios that rival traditional radioisotopic techniques while maintaining biological integrity of the receptor-ligand interaction. In addition, the ability to discriminate between active and inactive compounds in a mock galanin screen is demonstrated with low well-to-well variability, allowing reliable determination of positive hits even for low-affinity interactions.     

Regulation of plasmin activity by annexin II tetramer

Annexin II tetramer (AIIt) is a major Ca(2+)-binding protein of the endothelial cell surface which has been shown to stimulate the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. In the present report, we have examined the regulation of plasmin activity by AIIt. The incubation of plasmin with AIIt resulted in a 95% loss in plasmin activity. SDS-PAGE analysis established that AIIt stimulated the autoproteolytic digestion of plasmin heavy and light chains. The kinetics of AIIt-stimulated plasmin autoproteolysis were first-order, suggesting that binding of plasmin to AIIt resulted in the spontaneous autoproteolysis of the bound plasmin. AIIt did not affect the activity of other serine proteases such as t-PA or urokinase-type plasminogen activator. Furthermore, other annexins such as annexin I, II, V, or VI did not stimulate plasmin autoproteolysis. Increasing the concentration of AIIt on the surface of human 293 epithelial cells increased cell-mediated plasmin autoproteolysis. Thus, in addition to stimulating the formation of plasmin, AIIt also promotes plasmin inactivation. These results therefore suggest that AIIt may function to provide the cell surface with a transient pulse of plasmin activity.     

Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation

The catalytic domains of the pterin-dependent enzymes phenylalanine hydroxylase and tyrosine hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specificity, seven residues in the active site of phenylalanine hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the corresponding residues in tyrosine hydroxylase. Analysis of the effects of the mutations on the isolated catalytic domain of phenylalanine hydroxylase identified three residues that contribute to the ability to hydroxylate tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-length phenylalanine hydroxylase and the complementary mutations into tyrosine hydroxylase. The steady-state kinetic parameters of the mutated enzymes showed that the identity of the residue in tyrosine hydroxylase at the position corresponding to position 379 of phenylalanine hydroxylase is critical for dihydroxyphenylalanine formation. The relative specificity of tyrosine hydroxylase for phenylalanine versus tyrosine, as measured by the (V/K(phe))/(V/K(tyr)) value, increased by 80000-fold in the D425V enzyme. However, mutation of the corresponding valine 379 of phenylalanine hydroxylase to aspartate was not sufficient to allow phenylalanine hydroxylase to form dihydroxyphenylalanine at rates comparable to that of tyrosine hydroxylase. The double mutant V379D/H264Q PheH was the most active at tyrosine hydroxylation, showing a 3000-fold decrease in the (V/K(phe))/(V/K(tyr)) value.     

Tumor-enhancing effects of cholic acid are exerted on the early stages of colon carcinogenesis via induction of aberrant crypt foci with an enhanced growth phenotype.

The objective of the study was to establish whether cholic acid (CHA) enhanced colonic tumor incidence in the early phase of carcinogenesis. Male, Sprague-Dawley rats (n = 180) were injected twice with azoxymethane (AOM) (15 mg x kg(-1) body weight x week(-1), s.c., given 1 week apart). Following the first AOM injection, animals were randomly assigned to two groups, control AIN-93G diet (CON) or control diet containing 0.2% CHA by weight (CHA). Three weeks after the first injection, 20 animals (10 animals/group) were killed and aberrant crypt foci (ACF) were enumerated. The remaining animals were further subdivided and animals randomly assigned to CON or CHA diets, creating four treatments: CON-CON, CON-CHA, CHA-CHA, and CHA-CON. After 3, 12, and 20 weeks (following the first carcinogen injection), the animals were killed and the number and crypt multiplicity of ACF enumerated. Macroscopic tumors were evaluated at week 20. Total ACF were not different between groups. Average crypt multiplicity and medium (4-6 crypts/focus) and large (> or = 7 crypts/focus) ACF were greater in CHA-CHA and CHA-CON compared with CON-CON and CON-CHA (p < 0.01). Transient exposure to CHA (CHA-CON) was sufficient to induce development of ACF with an accelerated growth phenotype and elicit a tumor-enhancing effect. CHA-CHA had the highest tumor incidence (82.8%, p < 0.05) followed by CHA-CON (56.7%, p < 0.05), and tumor multiplicity and number of tumors per rat in CHA-CON were similar to CHA-CHA (2.29 and 1.3 versus 2.33 and 1.9, respectively). Delayed intervention with CHA (CON-CHA) produced a tumor outcome similar to CON-CON (31 and 30%, respectively), it did not enhance colonic tumor incidence. Taken collectively these results suggest CHA was effective in enhancing colon carcinogenesis during early phases and ineffective in post-initiation phases.     

Comparison of three methods of acute administration of progesterone on ovarian follicular development and the timing and synchrony of ovulation in Bos indicus heifers

The aim of this study was to induce the formation of a persistent dominant ovarian follicle and to compare the effects of 3 methods of acute administration of P4 on ovarian follicular development and on the timing and synchrony of ovulation. Stage of the estrous cycle was initially synchronized in Bos indicus heifers with a norgestomet implants (3 mg) for 10 d and with an analogue of PGF2 alpha (15 mg) on the first and last day of norgestomet treatment. Eight days after removal of the implants, heifers were randomly assigned to 4 groups. All heifers received a norgestomet implant (Day 0), which was removed 17 d later (Day 17); PGF2 alpha was administered on Days 0 and 4. Heifers in the control group (n = 5) received no other treatment. On Day 10 heifers in Group P4C (n = 5) were treated with a CIDR for 24 h; heifers in Group P4O (n = 5) were administered 100 mg i.m. of P4 in oil, while heifers in Group P4S (n = 5) were administered 100 mg i.m. of P4 in saline/alcohol. Data were analyzed using bootstrap estimates of location (mean) and spread (standard deviation; SD). Compared with the control heifers, day of emergence of the ovulatory follicle was delayed, and age and duration of dominance of the ovulatory follicle were reduced in the P4C and P4O heifers (P < 0.05) but not in the P4S heifers (P > 0.05). In all groups treated with P4 both the mean and variability (SD) in the timing of ovulation did not differ with that of the control group (P > 0.05) but there was less variability in the day of emergence, age, duration of dominance and diameter of the ovulatory follicle than in the control group (P < 0.05). Delayed timing and reduced synchrony (SD) of ovulation and greater age of the ovulatory follicle (P < 0.05) occurred in P4S heifers than in P4C heifers. We conclude that administration of 100 mg of P4 in oil is as effective as treatment with a CIDR for synchronizing emergence and ovulation of a newly recruited dominant follicle. However, reduced synchrony of ovulation, greater age of the ovulatory follicle and delayed timing of ovulation occurred following administration 100 mg of P4 in saline/alcohol compared with the CIDR device.     

CNS1 Encodes an Essential p60/Sti1 Homolog in Saccharomyces cerevisiae That Suppresses Cyclophilin 40 Mutations and Interacts with Hsp90

Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40 kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and contains two domains, an amino-terminal prolyl isomerase domain and a carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which has moderate identity to the yeast p60 homolog STI1, was isolated as a high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed, essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with Hsp90. In summary, our findings identify a novel component of the Hsp90 chaperone complex that shares function with cyclophilin 40 and provide evidence that there are functional differences between two conserved sets of Hsp90 binding proteins in yeast.     

The acute impact of continuous positive airway pressure on nasal resistance: a randomized controlled comparison.

Subjective nasal obstruction is common among users of continuous positive airway pressure (CPAP). The aim of this study was to measure the acute effect of CPAP on nasal resistance and nasal symptoms in awake normal subjects. Twenty-four healthy CPAP-naive adults [8 men, 16 women; mean age 30 yr (SD 14)] underwent a randomized controlled crossover study comparing nasal CPAP (8 cmH(2)O) for 6 h on one occasion and the control condition (nasal mask without CPAP) on the other. Nasal resistance measurements (posterior active rhinometry) before and after the test exposure were similar on both test days. Nasal resistance during CPAP exposure [2.04 cmH(2)O.l(-1).s (SD 0.72)] was significantly lower than that of the control [2.67 cmH(2)O.l(-1).s (SD 1.07)]: mean difference 0.66 cmH(2)O.l(-1).s, 95% confidence interval 0.19-1.13 cmH(2)O.l(-1).s. The gradient in pressure from CPAP mask to posterior naris during CPAP exposure varied from 1.6 to 2 cmH(2)O but was not significantly different between time points. Subjective nasal symptom scores and peak nasal inspiratory flow rates did not change significantly on either test day. We conclude that in awake CPAP-naive normal subjects, acute CPAP exposure is associated with a reduction in nasal resistance compared with the control condition, but it is not associated with an immediate post-CPAP change in subjective or objective nasal resistance.