Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin alpha

Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin alpha (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin alpha (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome cz.ccirf;tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H(2)O(2), catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.     

Origin and diffusion of mtDNA haplogroup X

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA) sequences belonging to haplogroup X and the survey of the haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and Africa revealed that haplogroup X is subdivided into two major branches, here defined as “X1” and “X2.” The first is restricted to the populations of North and East Africa and the Near East, whereas X2 encompasses all X mtDNAs from Europe, western and Central Asia, Siberia, and the great majority of the Near East, as well as some North African samples. Subhaplogroup X1 diversity indicates an early coalescence time, whereas X2 has apparently undergone a more recent population expansion in Eurasia, most likely around or after the last glacial maximum. It is notable that X2 includes the two complete Native American X sequences that constitute the distinctive X2a clade, a clade that lacks close relatives in the entire Old World, including Siberia. The position of X2a in the phylogenetic tree suggests an early split from the other X2 clades, likely at the very beginning of their expansion and spread from the Near East.     

Cis- and trans-acting influences on telomeric position effect in Drosophila melanogaster detected with a subterminal transgene

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.     

Social correlates of stress in adult males of the great gerbil,Rhombomys opimus,in years of high and low population densities

The great gerbil, Rhombomys opimus, is the most social species in the Gerbillinae. The social structure consists of family groups that occupy isolated systems of burrows consisting of one breeding male, from one to seven females, and juveniles. During a year of peak density and one of density decline, we studied the influence of group size, group composition, local density, and distance to the nearest groups on fecal corticosterone and testosterone concentrations in breeding males. We also examined the relationship of hormone concentrations to the survival of males during the summer drought between the spring and the fall. We found that males differed in concentrations of steroid hormones. Concentrations of testosterone were lower whereas those of corticosterone tended to be higher in a year of high population densities compared with higher testosterone and lower corticosterone in a year with a lower density. This finding suggests that stress may be greater in higher densities because of increased social contact. Stepwise regression analysis revealed a positive and significant influence of the number of adult females in a family group on concentrations of fecal corticosterone and testosterone in adult males. Concentrations of corticosterone were also significantly higher in males that disappeared from family groups between the spring and the fall compared with males still alive in family groups in the fall. There was no change in concentrations of testosterone. These results suggest that social interactions within large family groups may be an important source of stress for adult males.     

Localization versus function of Rab3 proteins. Evidence for a common regulatory role in controlling fusion

Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca(2+)-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca(2+)-triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Lüthi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca(2+)-independent exocytosis that correlated with the inhibition of regulated Ca(2+)-triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knock-out studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.     

Receptor methylation controls the magnitude of stimulus-response coupling in bacterial chemotaxis

Motile prokaryotes employ a chemoreceptor-kinase array to sense changes in the media and properly adjust their swimming behavior. This array is composed of a family of Type I membrane receptors, a histidine protein kinase (CheA), and an Src homology 3-like protein (CheW). Binding of an attractant to the chemoreceptors inhibits CheA, which results in decreased phosphorylation of the chemotaxis response regulator (CheY). Sensitivity of the system to stimuli is modulated by a protein methyltransferase (CheR) and a protein methylesterase (CheB) that catalyze the methylation and demethylation of specific glutamyl residues in the cytoplasmic domain of the receptors. One of the most fundamental unanswered questions concerning the bacterial chemotaxis mechanism is the quantitative relationship between ligand binding to receptors and CheA inhibition. We show that the receptor glutamyl modifications cause adaptation by changing the gain (magnitude amplification) between attractant binding and kinase inhibition without substantially affecting ligand binding affinity. The mechanism adjusts receptor sensitivity to background stimulus intensity over several orders of magnitude of attractant concentrations. The cooperative effects of ligand binding appear to be minimal with Hill coefficients for kinase inhibition less than 2, independent of the state of glutamyl modification.     

Some new aspects of 17alpha-estradiol metabolism in man

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.