Bayesian analysis of serial dilution assays

In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments.     

N-isonicotinoyl-(L)-4-aminophenylalanine derivatives as tight binding VLA-4 antagonists

A series of isonicotinoyl-(L)-aminophenylalanine derivatives was prepared and evaluated as VLA-4 antagonists. These compounds exhibit subnanomolar binding affinity to VLA-4 and significant off-rates. The interplay between off-rate, protein binding and pharmacokinetics is discussed.     

14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity

We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.     

Death assemblages of molluscs on St Martin's Flats, Isles of Scilly: a surrogate for regional biodiversity?

We test the hypothesis that the biodiversity of the death assemblage of shelled molluscs (gastropods and bivalves) on a single sandflat is representative of the biodiversity of the regional living fauna from the complete spectrum of habitat types, and can therefore be used as a surrogate for the purposes of rapid biodiversity assessment. Two biodiversity indices considered appropriate for such an assessment are average taxonomic distinctness (⁺) and the variation in taxonomic distinctness (⁺), which are, respectively, measures of the degree to which the species are taxonomically related to each other (i.e. the average taxonomic spread), and the degree to which taxa are over or under represented (i.e. the evenness of the distribution across the taxonomic tree). In each case, measured values were compared with the regional living species pool using a randomisation test. Collections and identifications of shells were made by both a non-expert in mollusc taxonomy and by taxonomic experts, the latter using microscopic examination of sediment samples as well as simple surface searches for macro-species. In all studies, ⁺ for the gastropods in the death assemblage was fully representative of the regional living fauna, but for bivalves it was significantly lower. This is because most of the bivalves were characteristic of the sandflat habitat, which introduced a bias, whereas post-mortem transport of gastropods, characterising exotic habitats, is more random. For gastropods, in all studies but one, ⁺ was significantly higher than expected from a random sample of the regional species pool, and for bivalves this was the case for all studies, suggesting the over-representation of some taxa and the under-representation of others. For the total shelled molluscan fauna (gastropods + bivalves) all ⁺ values were below, and all ⁺ values above, expectation. Results obtained by experts and a non-expert in mollusc taxonomy did not differ. In general, death assemblages at a single location are only likely to be representative of a wider region if there is randomisation as a result of post-mortem transport processes.     

Electroantennogram responses of male Sphinx perelegans hawkmoths to floral and ‘green-leaf volatiles’

The effects of sublethal dosages of the chloronicotinyl insecticide imidacloprid on different strains of the tobacco whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), have been studied after leaf dip and systemic application. All bioassays were performed with the insecticide susceptible strain, SUD-S, and two Spanish biotypes, ALM-2 and LMPA-2, both resistant to conventional insecticides and with a lower susceptibility towards imidacloprid. Honeydew, excreted by all strains feeding on treated and untreated cotton leaf discs was quantified by photometric analysis of its carbohydrate content. EC50-values for the depression of honeydew excretion in female adults after systemic application of imidacloprid were calculated at 0.037 ppm, 0.027 ppm and 0.048 ppm for strains SUD-S, ALM-2 and LMPA-2, respectively, indicating no significant differences between strains in feeding behaviour throughout an 48 h testing period. Depending on the strain these EC50-values were 150- to 850-times lower than LC50-values calculated for mortality in the same bioassay. Starvation tests revealed mean survival times of >48 h for female adults placed on agar without leaf discs, indicating that sublethal dosages of imidacloprid which caused antifeedant responses, were probably not covered in common 48 h systemic bioassays, used to monitor resistance to imidacloprid. Effects of sublethal dosages on honeydew excretion after leaf dip application seem to be minor. In choice situations with systemically treated and untreated leaf discs in a single container, female adults of B. tabaci showed a clear preference for the untreated leaf discs. However, when using leaf discs treated by painting the surface with imidacloprid in the same bioassay, feeding activities on treated and untreated leaf discs were not significantly different. The results of the present study demonstrate the antifeedant properties of imidacloprid on B. tabaci, which might play an essential role after soil application or seed treatment under field conditions.     

Congenital constriction band syndrome. Pathophysiology and treatment.

The clinical manifestations of 88 children with congenital constriction band syndrome involvement of the hand were reviewed. Seventy-five of these children had evidence of digital or limb amputations, with 235 upper limb amputations and 138 lower limb amputations. In the hand, digital amputations were most common in the index, middle, and ring fingers, whereas in the foot, amputations of the hallux were most often noted. Band indentation was often present at multiple levels. Proximal bands may be associated with neural compression. Syndactyly was invariably associated with a proximal interdigital sinus or cleft and was frequently associated with distal amputation. Examination of a 27-week gestation stillborn specimen having manifestations of congenital constriction band syndrome demonstrated the intrauterine biologic response to band constriction. The variable clinical manifestations of congenital constriction band syndrome can best be explained as the response of the growing, embryologically defined limb to intrauterine deformation or band-induced compression and ischemia.     

Measurements of the molecular size of the simian virus 40 large T antigen.

A measure of the molecular weight of the large simian virus 40 T antigen was sought by SDS-polyacrylamide gel electrophoresis, random-coil chromatography, and sedimentation-velocity analysis in a density gradient. Large T antigen obtained from a simian virus 40-transformed human cell line either by immunoprecipitation or by standard preparatory methods migrated like a 94,000-molecular-weight (approximately 94K) polypeptide in SDS-gels but was found to have an approximate was observed with T antigen obtained from lytically infected monkey cells. In view of the strong theoretical basis for the guanidine method and the agreement with the sedimentation data, these findings suggest that the molecular weight of this protein is approximately 75 to 80K as opposed to 94 to 100K and, therefore, that considerably less than the entire early region of simian virus 40 is required to encode it. This size estimate is in keeping with earlier results which revealed a normal-size T antigen in cells infected with viable deletion mutants lacking as much as 10% of the early region.     

Acetylation of 13-sophorosyloxydocosanoic acid by an acetyltransferase purified from Candida bogoriensis.

Acetyl-coenzyme A: 13-sophorosyloxydocosanoic acid (Glc2HDA) acetyltransferase was purified 14-fold in low yield from Candida bogoriensis cells. The enzyme catalyzes acetylation of the 6' and 6" positions of the sophorosyl group, producing the 13-[2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyloxy]-docosanoic acid 6',6"-diacetate (Ac2Glc2HDA) and monoacetate (AcGlc2HDA) in a product ratio of 5:1. Neither the purification steps nor heat denaturation studies indicated separation of the first and second acetylation steps. The acetyltransferase has a molecular weight of about 500,000 as determined by gel filtration on a Sepharose 4-B column. It shows a pH optimum range from 7 to 9, is strongly inhibited by 1 mM concentrations of the sulfhydryl reagents N-ethylmaleimide, p-hydroxymercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid), but only partly inhibited by 10 mM iodoacetamide. It has an apparent Km of 30 muM for acetyl-CoA, utilizes propionyl-CoA at 45% the rate of acetyl-CoA, and utilizes longer chain acyl-CoA derivatives much less efficiently. The critical micelle concentrations of the C. bogoriensis glycolipids in pH 7.7 phosphate buffer were estimated by pinacyanol chloride binding as follows: Glc2HDA, 50 mum; AcGlc2HDA, 30 muM; Ac2Glc2HDA, 12 muM. The Stokes radius of Ac2Glc2HDA micelles was 22 A as estimated by gel filtration on Bio-Gel P-150. Glc2HDA was a much better acceptor than its methyl ester in the acetyltransferase assay. A plateau in the Glc2HDA saturation curve at 50 muM and a corresponding break in the reciprocal plot at this concentration indicate the enzyme utilizes the monomeric form of this lipid as substrate.     

Use of Firefly Luciferase for ATP Measurement: Other Nucleotides Enhance Turnover

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (>8μM) or a fairly constant production of light with lower concentrations of ATP (< 1 μM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.