Review paper: origin and molecular pathology of adrenocortical neoplasms.

Neoplastic adrenocortical lesions are common in humans and several species of domestic animals. Although there are unanswered questions about the origin and evolution of adrenocortical neoplasms, analysis of human tumor specimens and animal models indicates that adrenocortical tumorigenesis involves both genetic and epigenetic alterations. Chromosomal changes accumulate during tumor progression, and aberrant telomere function is one of the key mechanisms underlying chromosome instability during this process. Epigenetic changes serve to expand the size of the uncommitted adrenal progenitor population, modulate their phenotypic plasticity (i.e., responsiveness to extracellular signals), and increase the likelihood of subsequent genetic alterations. Analyses of heritable and spontaneous types of human adrenocortical tumors documented alterations in either cell surface receptors or their downstream effectors that impact neoplastic transformation. Many of the mutations associated with benign human adrenocortical tumors result in dysregulated cyclic adenosine monophosphate signaling, whereas key factors and/or signaling pathways associated with adrenocortical carcinomas include dysregulated expression of the IGF2 gene cluster, activation of the Wnt/beta-catenin pathway, and inactivation of the p53 tumor suppressor. A better understanding of the factors and signaling pathways involved in adrenal tumorigenesis is necessary to develop targeted pharmacologic and genetic therapies.

Cytochrome b5 expression in gonadectomy-induced adrenocortical neoplasms of the domestic ferret (Mustela putorius furo).

Whereas the adrenal glands of healthy ferrets produce only limited amounts of androgenic steroids, adrenocortical neoplasms that arise in neutered ferrets typically secrete androgens or their derivative, estrogen. The 17,20-lyase activity of cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) must increase to permit androgen biosynthesis in neoplastic adrenal tissue. We screened ferret adrenocortical tumor specimens for expression of cytochrome b(5) (cyt b(5)), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17. Cyt b(5) immunoreactivity was evident in 24 of 25 (96%) adrenocortical adenomas/carcinomas from ferrets with signs of ectopic sex steroid production. Normal adrenocortical cells lacked cyt b(5), which may account for the low production of adrenal androgens in healthy ferrets. Other markers characteristic of gonadal somatic cells, such as luteinizing hormone receptor, aromatase, and GATA4, were coexpressed with cyt b(5) in some of the tumors. We concluded that cyt b(5) is upregulated during gonadectomy-induced adrenocortical neoplasia and is a marker of androgen synthetic potential in these tumors.

Molecular cloning, characterization, and expression studies of water buffalo (Bubalus bubalis) somatotropin.

Cloning, high-level expression, and characterization of the somatotropin (ST) gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST) are described. Coding, non-coding, and promoter regions of BbST were amplified and sequenced. Sequence analysis revealed several silent and two interesting point mutations on comparison with STs of other vertebrate species. One interesting variation in the BbST sequence was the replacement of a conserved glutamine residue by arginine. A plasmid was also constructed for the production of BbST in Escherichia coli BL21 (RIPL) CodonPlus, under the control of IPTG-inducible T7-lac promoter. High-level expression could be obtained by synthesizing a codon-optimized ST gene and expressing it in the form of inclusion bodies. The inclusion bodies represented over 20% of the E. coli cellular proteins. The biologically active conformation of purified BbST was confirmed by its efficient growth promoting activity in Nb2 cell proliferation assay. The expression system and purification strategy employed promise to be a useful approach to produce BbST for further use in structure-function studies and livestock industry.

GATA-4 is a granulosa cell factor employed in inhibin-alpha activation by the TGF-beta pathway.

Part of heterodimeric inhibin, inhibin-alpha is crucial for mammalian ovarian function. Regulation of inhibin-alpha expression in granulosa cells is both endocrine, primarily by follicle-stimulating hormone (FSH), and paracrine, primarily by members of the transforming growth factor beta (TGF-beta) superfamily. Smad proteins transmit TGF-beta signals to the nucleus, but the cooperating transcription factors involved in inhibin-alpha promoter activation remain unknown. Transcription factor GATA-4 regulates inhibin-alpha in gonadal cells, and the FSH cascade activates GATA-4. We hypothesized that the TGF-beta signalling cascade and GATA-4 also cooperate to regulate inhibin-alpha expression. In KK-1 granulosa tumour cells, which resemble normal granulosa cells and express inhibin-alpha, we found that TGF-beta upregulated GATA-4 expression. Transient transfection experiments in KK-1 cells demonstrated that dominant negative GATA-4 variants or mutations of GATA-binding sites in the inhibin-alpha promoter attenuated TGF-beta-induced gene activation. In GATA-4-deficient COS-7 cells, TGF-beta enhanced the expression of the inhibin-alpha promoter only in the presence of exogenous GATA-4. Smad3, but not Smad2, cooperated with GATA-4 in the transcriptional activation of the inhibin-alpha promoter, and immunoprecipitation experiments in KK-1 cells revealed a physical Smad3:GATA-4 interaction. Our data suggest that GATA-4, interacting with Smad3, is a cofactor for TGF-beta signalling to activate inhibin-alpha in granulosa cells.

Gonadectomy-induced adrenocortical neoplasia in the domestic ferret (Mustela putorius furo) and laboratory mouse.

Sex steroid-producing adrenocortical adenomas and carcinomas occur frequently in neutered ferrets, but the molecular events underlying tumor development are not well understood. Prepubertal gonadectomy elicits similar tumors in certain inbred or genetically engineered strains of mice, and these mouse models shed light on tumorigenesis in ferrets. In mice and ferrets, the neoplastic adrenocortical cells, which functionally resemble gonadal steroidogenic cells, arise from progenitors in the subcapsular or juxtamedullary region. Tumorigenesis in mice is influenced by the inherent susceptibility of adrenal tissue to gonadectomy-induced hormonal changes. The chronic elevation in circulating luteinizing hormone that follows ovariectomy or orchiectomy is a prerequisite for neoplastic transformation. Gonadectomy alters the plasma or local concentrations of steroid hormones and other factors that affect adrenocortical tumor development, including inhibins, activins, and Müllerian inhibiting substance. GATA-4 immunoreactivity is a hallmark of neoplastic transformation, and this transcription factor might serve to integrate intracellular signals evoked by different hormones. Synergistic interactions among GATA-4, steroidogenic factor-1, and other transcription factors enhance expression of inhibin-alpha and genes critical for ectopic sex steroid production, such as cytochrome P450 17alpha-hydroxylase/17,20 lyase and aromatase. Cases of human adrenocortical neoplasia have been linked to precocious expression of hormone receptors and to mutations that alter the activity of G-proteins or downstream effectors. Whether such genetic changes contribute to tissue susceptibility to neoplasia in neutered ferrets and mice awaits further study.

Modelling nitrogen saturation and carbon accumulation in heathland soils under elevated nitrogen deposition.

A simple model of nitrogen (N) saturation, based on an extension of the biogeochemical model MAGIC, has been tested at two long-running heathland N manipulation experiments. The model simulates N immobilisation as a function of organic soil C/N ratio, but permits a proportion of immobilised N to be accompanied by accumulation of soil carbon (C), slowing the rate of C/N ratio change and subsequent N saturation. The model successfully reproduced observed treatment effects on soil C and N, and inorganic N leaching, for both sites. At the C-rich upland site, N addition led to relatively small reductions in soil C/N, low inorganic N leaching, and a substantial increase in organic soil C. At the C-poor lowland site, soil C/N ratio decreases and N leaching increases were much more dramatic, and soil C accumulation predicted to be smaller. The study suggests that (i) a simple model can effectively simulate observed changes in soil and leachate N; (ii) previous model predictions based on a constant soil C pool may overpredict future N leaching; (iii) N saturation may develop most rapidly in dry, organic-poor, high-decomposition systems; and (iv) N deposition may lead to significantly enhanced soil C sequestration, particularly in wet, nutrient-poor, organic-rich systems.

Dyskeratosis congenita -- a disease of dysfunctional telomere maintenance.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome associated with abnormalities of the skin, fingernails, and tongue. Other clinical manifestations may include epiphora, lung fibrosis, liver cirrhosis, osteoporosis, and a predisposition to develop a variety of malignancies. The clinical picture often resembles that of a premature aging syndrome and tissues affected are those with a high cell turnover. DC has been linked to mutations in at least four distinct genes, three of which have been identified. The product of these genes, dyskerin, the telomerase RNA (TERC), and the catalytic unit of telomerase (TERT) are part of a ribonucleoprotein complex, the telomerase enzyme, that is essential for the elongation and maintenance of chromosome ends or telomeres. All patients with DC have excessively short telomeres, indicating that the underlying defect in these individuals is an inability to maintain the telomeres. The purpose of the current review is to highlight recent insights into the molecular pathogenesis of DC. We discuss the impact these findings have on our current understanding of telomere function and maintenance, and on the diagnosis, management, and treatment of patients with conditions caused by dysfunctional telomeres.

Transcription factor GATA-4 is a marker of anaplasia in adrenocortical neoplasms of the domestic ferret (Mustela putorius furo).

Adrenocortical neoplasms are a common cause of morbidity in neutered ferrets. Recently we showed that gonadectomized DBA/2J mice develop adrenocortical tumors that express transcription factor GATA-4. Therefore, we screened archival specimens of adrenocortical neoplasms from neutered ferrets to determine whether GATA-4 could be used as a tumor marker in this species. Nuclear immunoreactivity for GATA-4 was evident in 19/22 (86%) of ferret adrenocortical carcinomas and was prominent in areas exhibiting myxoid differentiation. Normal adrenocortical cells lacked GATA-4 expression. Two other markers of adrenocortical tumors in gonadectomized mice, inhibin-alpha and luteinizing hormone receptor, were coexpressed with GATA-4 in some of the ferret tumors. No GATA-4 expression was observed in three cases of nodular hyperplasia, but patches of anaplastic cells expressing GATA-4 were evident in 7/14 (50%) of tumors classified as adenomas. We conclude that GATA-4 can function as a marker of anaplasia in ferret adrenocortical tumors.

A modular minimal cell model: purine and pyrimidine transport and metabolism.

A more complete understanding of the relationship of cell physiology to genomic structure is desirable. Because of the intrinsic complexity of biological organisms, only the simplest cells will allow complete definition of all components and their interactions. The theoretical and experimental construction of a minimal cell has been suggested as a tool to develop such an understanding. Our ultimate goal is to convert a "coarse-grain" lumped parameter computer model of Escherichia coli into a genetically and chemically detailed model of a "minimal cell." The base E. coli model has been converted into a generalized model of a heterotrophic bacterium. This coarse-grain minimal cell model is functionally complete, with growth rate, composition, division, and changes in cell morphology as natural outputs from dynamic simulations where only the initial composition of the cell and of the medium are specified. A coarse-grain model uses pseudochemical species (or modules) that are aggregates of distinct chemical species that share similar chemistry and metabolic dynamics. This model provides a framework in which these modules can be "delumped" into chemical and genetic descriptions while maintaining connectivity to all other functional elements. Here we demonstrate that a detailed description of nucleotide precursors transport and metabolism is successfully integrated into the whole-cell model. This nucleotide submodel requires fewer (12) genes than other theoretical predictions in minimal cells. The demonstration of modularity suggests the possibility of developing modules in parallel and recombining them into a fully functional chemically and genetically detailed model of a prokaryote cell.

Nude mice as a model for gonadotropin-induced adrenocortical neoplasia.

Certain inbred mice (e.g., DBA/2J, CE) develop sex steroid producing adrenocortical tumors following gonadectomy. This adrenal response is thought to result from an unopposed increase in circulating gonadotropins and/or a decrease in factor(s) of gonadal origin. To differentiate between these two possibilities, we utilized the NU/J strain of nude mice, which are immunologically compromised and therefore permissive to xenografts. One group of female nude mice was gonadectomized, while another group of females received xenografts of CHO cells stably transfected with human chorionic gonadotropin (hCG). After 1-2 months, subcapsular adrenocortical neoplasms containing sex steroid-producing cells were observed in both groups. We conclude that high levels of circulating gonadotropins are sufficient to induce adrenocortical tumorigenesis, even in the presence of intact gonads.

Corn fiber hydrolysis by Thermobifida fusca extracellular enzymes.

Thermobifida fusca was grown on cellulose (Solka-Floc), xylan or corn fiber and the supernatant extracellular enzymes were concentrated. SDS gels showed markedly different protein patterns for the three different carbon sources. Activity assays on a variety of synthetic and natural substrates showed major differences in the concentrated extracellular enzyme activities. These crude enzyme preparations were used to hydrolyze corn fiber, a low-value biomass byproduct of the wet milling of corn. Approximately 180 mg of reducing sugar were produced per gram of untreated corn fiber. When corn fiber was pretreated with alkaline hydrogen peroxide, up to 429 mg of reducing sugars were released per gram of corn fiber. Saccharification was enhanced by the addition of beta-glucosidase or by the addition of a crude xylanase preparation from Aureobasidium sp.

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2).

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.

Transcription factors GATA-4 and GATA-6 during mouse and human adrenocortical development.

Our earlier work implicates transcription factors GATA-4 and GATA-6 in the murine adrenal. We have now studied their expression during mouse and human adrenal development in detail. GATA-4 and GATA-6 mRNAs are readily detectable from embryonic day 15 in mouse and gestational week 19 in human adrenal cortex. In postnatal adrenal, GATA-4 expression is down-regulated, whereas GATA-6 mRNA and protein continue to be abundantly present. In a human adrenocortical cell line NCI-H295R, GATA-6 mRNA is up-regulated by cAMP. This cell line does not express GATA-4. Our findings suggest that GATA-6 expression is hormonally controlled, and required throughout adrenal development from fetal to adult age. GATA-4, on the other hand, may serve a role in fetal adrenal gene regulation.

Use of remifentanil in a patient with peripartum cardiomyopathy requiring Caesarean section.

We describe a case of a 26 yr old primigravida at 39 weeks' gestation, with a diagnosis of peripartum cardiomyopathy, requiring urgent Caesarean section. The patient presented in severe heart failure and active labour. A general anaesthetic, using a target-controlled infusion of propofol and an intravenous infusion of remifentanil, was used to provide stable anaesthesia and analgesia for a successful delivery. The unusual diagnosis of peripartum cardiomyopathy and the potential benefits of the use of remifentanil in high-risk obstetric surgery are discussed.

A selective inhibitor of Escherichia coli prephenate dehydratase.

To identify selective prephenate dehydratase (PDT) inhibitors, a series of substituted biphenic acid derivatives was synthesized using the Ullmann reaction. Screening experiments identified 18 as a promising new PDT inhibitor.

Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli.

Genetically engineered E. coli, which express both a Hg2+ transport system and metallothionein, were tested for their ability to remove mercury from wastewater. The wastewater contained more than ten different ions, including 2.58 mg/l mercury, and its pH was 9.6. Mercury uptake was faster from the wastewater than from distilled water, probably because of the higher ionic strength, as the high pH had little effect on mercury accumulation. EDTA also stimulated mercury uptake rather than inhibiting it. A hollow-fiber bioreactor was used to retain induced cells for continuous mercury uptake. The cells removed more than 99% of the mercury in the wastewater and the final amount of mercury accumulated was 26.8 mg/g cell dry weight, while none of the other ions were removed from the water. These results indicated that the induced cells had a high affinity and specificity for mercury.

The way in which intervention studies have "personality" and why it is important to meta-analysis.

Intervention studies represent webs of interrelated substantive and methodological characteristics that take on different patterns in different studies and different intervention areas. All too often, meta-analysts do not give close attention to the possibility that these interrelated differences among studies are related in complex ways to study effect sizes and, consequently, run considerable risk of reporting results that are misleading or flatly wrong. To remedy this situation, improvements are needed in both the method and practice of meta-analysis so that greater attention can be given to effect size variation, the generalizability of study results, and the systematic multivariate relationships between study characteristics and the effect sizes reported in those studies.

Cloning and biochemical characterization of BglC, a beta-glucosidase from the cellulolytic actinomycete Thermobifida fusca.

An operon, bglABC, that encodes two sugar permeases and a beta-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular beta-glucosidase (beta-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both beta 1 --> 4 and beta 1 --> 2 glycosidic bonds, and was most active against cellobiose (Vmax = 29, Km = 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-beta-glucosidase activity on p-nitrophenyl-beta-d-glucopyranoside and p-nitrophenyl-beta-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum of 50 degrees C. The enzyme was stable at 60 degrees C, but was rapidly inactivated at 65 degrees C. BglC was inhibited by low concentrations of gluconolactone, but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are well suited for use in a process to hydrolyze biomass cellulose to glucose.

Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet beta cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528-539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC(50) > 100 microM), a larger 16-mer fragment, 526-541, shows activity in the low micromolar range (EC(50) = 2.3 microM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.