Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for CA-binding protein (GABP)

Within steroid receptor heterocomplexes the large tetraticopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (HspSO) and act coordinately with HspSO to modulate receptor activity. The reversible nature of the interaction between the immunophilins and HspSO suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a fi-kilobase (kb) 5 ' -flanking region of the human gene and demonstrated that a similar to 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GAFF is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Model studies on the role of citrate, malate and pectin esterification on the enzymatic degradation of Al- and Ca-pectate gels: possible implications for Al-tolerance

Aluminium (At) tolerance in plants may be conferred by reduced binding of Al in the cell wall through low root cation exchange capacity (CEC) or by organic acid exudation. Root CEC is related to the degree of esterification (DE) of pectin in the cell wall, and pectin hydrolysis plays a role in cell expansion. Therefore, it was hypothesised that Al-tolerant plants with a low root CEC maintain pectin hydrolysis in the presence of Al, allowing cell expansion to continue. Irrespective of the DE, binding of Al to pectin reduced the enzymatic hydrolysis of Al-pectin gels by polygalacturonase (E.C. 3.2.1.15). Pectin gels with calcium (Ca) were slightly hydrolysed by polygalacturonase. It was concluded, therefore, that Al tolerance conferred by low root CEC is not mediated by the ability to maintain pectin hydrolysis. Citrate and malate, but not acetate, effectively dissolved Al-pectate gel and led to hydrolysis of the dissolved pectin by polygalacturonase. The organic acids did not dissolve Ca-pectate, nor did they increase pectin hydrolysis by polygalacturonase. It was concluded that exudation of some organic acids can remove Al bound to pectin and this could alleviate toxicity, constituting a tolerance mechanism. (C) 2003 Editions scientitiques et medicales Elsevier SAS. All rights reserved.

A review of the use of the basic cation saturation ratio and the "ideal" soil

The use of 'balanced' Ca, Mg, and K ratios, as prescribed by the basic cation saturation ratio (BCSR) concept, is still used by some private soil-testing laboratories for the interpretation of soil analytical data. This review aims to examine the suitability of the BCSR concept as a method for the interpretation of soil analytical data. According to the BCSR concept, maximum plant growth will be achieved only when the soil’s exchangeable Ca, Mg, and K concentrations are approximately 65 % Ca, 10 % Mg, and 5 % K (termed the ‘ideal soil’). This ‘ideal soil’ was originally proposed by Firman Bear and co-workers in New Jersey (USA) during the 1940s as a method of reducing luxury K uptake by alfalfa (Medicago sativa L.). At about the same time, William Albrecht, working in Missouri (USA), concluded through his own investigations that plants require a soil with a high Ca saturation for optimal growth. Whilst it now appears that several of Albrecht’s experiments were fundamentally flawed, the BCSR (‘balanced soil’) concept has been widely promoted, suggesting that the prescribed cationic ratios provide optimum chemical, physical, and biological soil properties. Our examination of data from numerous studies (particularly those of Albrecht and Bear, themselves) would suggest that, within the ranges commonly found in soils, the chemical, physical, and biological fertility of a soil is generally not influenced by the ratios of Ca, Mg, and K. The data do not support the claims of the BCSR, and continued promotion of the BCSR will result in the inefficient use of resources in agriculture and horticulture.

Synthesis, structure and magnetism of the oxalato-bridged chromium(III) complex [NBu4n](4)[Cr-2(ox)(5)]center dot 2CHCl(3)

The binuclear complex [NBu4n](4)[Cr-2(ox)(5)]. 2CHCl(3) has been prepared by an ion-exchange procedure employing Dowex 50WX2 cation-exchange resin in the n-butylammonium form and potassium tris(oxalato)chromate(III). The dimeric complex was characterised by a crystal structure determination: monoclinic, space group C2/c, a = 29.241(7), b = 15.192(2), c = 22.026(5) Angstrom, beta = 94.07(1)degrees, Z = 4. The magnetic susceptibility (300-4.2 K) indicated that the chromium(III) sites were antiferromagnetically coupled (J = -3.1 cm(-1)).

Anatomy of the mouthparts and digestive tract during feeding in larvae of the parasitoid wasp Trichogramma australicum Girault (Hymenoptera : Trichogrammatidae)

To aid in the development of artificial diets for mass rearing parasitioids, we investigated the anatomical changes in the digestive tract during feeding behaviour of larval Trichogramma australicum (Hymenoptera: Trichogrammatidae). Larvae begin to feed immediately upon eclosion and feed continuously for 4 h until replete. Feeding is characterised by rhythmic muscle contractions (ca 1 per s) of the pharynx. Contractions of the pharyngeal dilator muscles lift the roof of the lobe-shaped pharynx away from the floor of the chamber, opening the mouth and pumping food into the pharyngeal cavity. Another muscle contraction occurs about 0.5 s later, forcing the bolus of food through the oesophagus and into the midgut. The junction of fore- and midgut is marked by a cardiac valve. The midgut occupies most of the body cavity and is lined with highly vacuolated, flattened cells and a dispersed layer of muscle cells. In the centre of the midgut, food has the appearance of host egg contents. Food near the midgut epithelial cells has a finer, more homogeneous appearance. This change in the physical properties of the gut contents is indicative of the digestion process. In the prepupa, where digestion is complete, the entire gut contents have this appearance. After eclosion, the vitelline membrane remains attached to the posterior end of the larva. We believe this attachment to be adaptive in two ways: (1) to anchor the larva against the movements of its anterior portion, thereby increasing the efficiency of foraging within the egg, and (2) to prevent a free-floating membrane from clogging the mouthparts during ingestion. 1998 Elsevier Science Ltd. All rights reserved.

Likelihood-based estimation of the regression model with scrambled responses

A significant problem in the collection of responses to potentially sensitive questions, such as relating to illegal, immoral or embarrassing activities, is non-sampling error due to refusal to respond or false responses. Eichhorn & Hayre (1983) suggested the use of scrambled responses to reduce this form of bias. This paper considers a linear regression model in which the dependent variable is unobserved but for which the sum or product with a scrambling random variable of known distribution, is known. The performance of two likelihood-based estimators is investigated, namely of a Bayesian estimator achieved through a Markov chain Monte Carlo (MCMC) sampling scheme, and a classical maximum-likelihood estimator. These two estimators and an estimator suggested by Singh, Joarder & King (1996) are compared. Monte Carlo results show that the Bayesian estimator outperforms the classical estimators in almost all cases, and the relative performance of the Bayesian estimator improves as the responses become more scrambled.

Familial hyperaldosteronism type II: Description of a large kindred and exclusion of the aldosterone synthase (CYP11B2) gene

Familial hyperaldosteronism type II (FH-II) is characterized by autosomal dominant inheritance and hypersecretion of aldosterone due to adrenocortical hyperplasia or an aldosterone-producing adenoma; unlike FH type I (FH-I), hyperaldosteronism in FH-II is not suppressible by dexamethasone. Of a total of 17 FH-II families with 44 affected members, we studied a large kindred with 7 affected members that was informative for linkage analysis. Family members were screened with the aldosterone/PRA ratio test; patients with aldosterone/PRA ratio greater than 25 underwent fludrocortisone/salt suppression testing for confirmation of autonomous aldosterone secretion. Postural testing, adrenal gland imaging, and adrenal venous sampling were also performed. Individuals affected by FH-II demonstrated lack of suppression of plasma A levels after 4 days of dexamethasone treatment (0.5 mg every 6 h). All patients had neg ative genetic testing for the defect associated with FH-I, the CYP11B1/CYP11B2 hybrid gene. Genetic linkage was then examined between FH-II and aldosterone synthase (the CYP11B2 gene) on chromosome 8q. A polyadenylase repeat within the 5'-region of the CYP11B2 gene and 9 other markers covering an approximately 80-centimorgan area on chromosome 8q21-8qtel were genotyped and analyzed for linkage. Two-point logarithm of odds scores were negative and ranged from -12.6 for the CYP11B2 polymorphic marker to -0.98 for the D8S527 marker at a recombination distance (theta) of 0. Multipoint logarithm of odds score analysis confirmed the exclusion of the chromosome 8q21-8qtel area as a region harboring the candidate gene for FH-II in this family. We conclude that FH-II shares autosomal dominant inheritance and hyperaldosteronism with FH-I, but, as demonstrated by the large kindred investigated in this report, it is clinically and genetically distinct. Linkage analysis demonstrated that the CYP11B2 gene is not responsible for FH-II in this family; furthermore, chromosome 8q21-8qtel most likely does not harbor the genetic defect in this kindred.

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-H-3]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin I. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin I using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an cc-helical secondary structure. (C) 1998 Elsevier Science B.V, All rights reserved.

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gets. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE(22-88) fragment forms hetero-oligomers with MinE(+) when the proteins are co-expressed. In contrast, the MinE(36-88) fragment does not form MinE(+)/MinE(36-88) hetero-oligomers, although MinE36-88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE(+) in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of mini-celling by expression of MinE(36-88) in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE(36-88),nd the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Involvement of the N-finger in the self-association of GATA-1

Zinc fingers are recognized as small protein domains that bind to specific DNA sequences. Recently however, zinc fingers from a number of proteins, in particular the GATA family of transcription factors, have also been implicated in specific protein-protein interactions. The erythroid protein GATA-1 contains two zinc fingers: the C-finger, which is sufficient for sequence-specific DNA-binding, and the N-finger, which appears both to modulate DNA-binding and to interact with other transcription factors. We have expressed and purified the N-finger domain and investigated its involvement in the self-association of GATA-1. We demonstrate that this domain does not homodimerize but instead makes intermolecular contacts with the C-finger, suggesting that GATA dimers are maintained by reciprocal N-finger-C-finger contacts. Deletion analysis identifies a 25-residue region, C-terminal to the core N-finger domain, that is sufficient for interaction with intact GATA-1. A similar subdomain exists C-terminal to the C-finger, and we show that self-association is substantially reduced when both subdomains are disrupted by mutation. Moreover, mutations that impair GATA-1 self-association also interfere with its ability to activate transcription in transfection studies.

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional H-1/N-15 NMR spectroscopy, and we use H-1/N-15 spin relation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C-4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NE Notably, this face is well removed from the putative DNA-binding face of the NE an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.