Larry J. Bischof

An Endocrine-Exocrine Switch in the Activity of the Pancreatic Homeodomain Protein PDX1 through Formation of a Trimeric Complex with PBX1b and MRG1 (MEIS2)

ABSTRACT HOX proteins and some orphan homeodomain proteins form complexes with either PBX or MEIS subclasses of homeodomain proteins. This interaction can increase the binding specificity and transcriptional effectiveness of the HOX partner. Here we show that specific members of both PBX and MEIS subclasses form a multimeric complex with the pancreatic homeodomain protein PDX1 and switch the nature of its transcriptional activity. The two activities of PDX1 are exhibited through the 10-bp B element of the transcriptional enhancer of the pancreatic elastase I gene (ELA1). In pancreatic acinar cells the activity of the B element requires other elements of the ELA1 enhancer; in β-cells the B element can activate a promoter in the absence of other enhancer elements. In acinar cell lines the activity is mediated by a complex comprising PDX1, PBX1b, and MRG1 (MEIS2). In contrast, β-cell lines are devoid of PBX1b and MRG1, so that a trimeric complex does not form, and the β-cell-type activity is mediated by PDX1 without PBX1b and MRG1. The presence of specific nuclear isoforms of PBX and MEIS is precisely regulated in a cell-type-specific manner. The β-cell-type activity can be detected in acinar cells if the B element is altered to retain binding of PDX1 but prevent binding of the PDX1-PBX1b-MRG1 complex. These observations suggest that association with PBX and MEIS partners controls the nature of the transcriptional activity of the organ-specific PDX1 transcription factor in exocrine versus endocrine cells.

Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo

Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum

Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use.

Cloning and Characterization of the Human and Rat Islet-specific Glucose-6-phosphatase Catalytic Subunit-related Protein (IGRP) Genes

Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and ratIGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRPgene has five exons of similar length and coding sequence to the mouseIGRP gene and is located on human chromosome 2q28–32 adjacent to the myosin heavy chain 1B gene. In contrast, the ratIGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissue-restricted expression of theIGRP gene and the identification of key amino acid sequences that determine its biological activity.

Assays for toxicity studies in C. elegans with Bt crystal proteins.

Caenorhabditis elegans is well suited for toxicological studies owing to its established biology, short generation time, large brood size, and readily scorable life traits. Quantitative parameters of C. elegans that can be assayed include growth, size, progeny production, behavior, and mortality. Qualitative parameters of toxicity, such as changes in appearance or movement, can also be determined. This chapter describes four assays we have used for analyzing the toxic effects of Bacillus thuringiensis crystal proteins toward C. elegans. The assays are quantitative growth measurement, brood size measurement, and determination of lethal concentration, as well as a qualitative health assay based on worm appearance. Although these assays are described for crystal proteins, the approaches are suited for the studies of other toxins with C. elegans.

Cytochromes P450 12: diversity of ACTH (cAMP)-dependent transcription of bovine steroid hydroxylase genes.

An essential role of ACTH is to assure that optimal steroidogenic capacity is maintained in the adrenal cortex throughout life. This is achieved by maintaining transcriptional pressure on the genes encoding the adrenocortical steroid hydroxylases via the second messenger, cAMP. Even though these genes respond coordinately to cAMP, it has been surprising to discover that each gene uses its own unique cAMP response system during this coordinate response. Thus, different cis elements and sets of transcription factors control the cAMP responsiveness of each different steroid hydroxylase gene. Although the physiological basis of this diversity in biochemical mechanisms of transcriptional regulation is not apparent, a portion of this signaling pathway is common to all of these genes. In particular, the action of cAMP‐dependent protein kinase and an as yet uncharacterized cycloheximide‐sensitive step are necessary for ACTH‐mediated transcription of each gene. Biochemical characterization of these common steps in the ACTH‐dependent signaling pathways is essential to an understanding of the maintenance of optimal steroidogenic capacity in the adrenal cortex.—Waterman, M. R., Bischof, L. J. Diversity of ACTH (cAMP)‐dependent transcription of bovine steroid hydroxylase genes. FASEB J. 11, 419–427 (1997)

Pore worms: using Caenorhabditis elegans to study how bacterial toxins interact with their target host.

The interaction of pathogenic bacteria with a target host is regulated both by bacterial virulence factors and by host components that either protect the host or that promote pathogenesis. The soil nematode Caenorhabditis elegans is a host for a number of bacterial pathogens, as briefly reviewed here. Bacillus thuringiensis (Bt) is a pathogenic bacteria that C. elegans is likely to encounter naturally in the soil. The pore-forming Crystal (Cry) toxins made by Bt are recognized as the dominant virulence factor in this host-pathogen interaction. Forward genetic screens for C. elegans mutants resistant to the Cry toxin, Cry5B, have identified a host carbohydrate structure that promotes pathogenesis. Data suggest this structure is likely to be a Cry5B receptor expressed in the host intestine. This finding is discussed in light of other carbohydrate receptors for bacterial toxins. To investigate host-toxin interactions on a global level, the response of C. elegans to the pore-forming Cry5B is also being investigated by gene transcription profiling (microarrays). These data are beginning to reveal a diverse intracellular response to toxin exposure. To put these investigations in perspective, host responses to other pore-forming toxins are discussed. Investigations with Cry5B in C. elegans show a promising beginning in helping to elucidate host-toxin and host-pathogen interactions.

Resistance to Bacillus thuringiensis Toxin in Caenorhabditis elegans from Loss of Fucose

A mutation in the Caenorhabditis elegans bre-1 gene was isolated in a screen for Bacillus thuringiensis toxin-resistant (bre) mutants to the Cry5B crystal toxin made by B. thuringiensis. bre-1 mutant animals are different from the four other cloned bre mutants in that their level of resistance is noticeably lower. bre-1 animals also display a significantly reduced brood size at 25 °C. Here we cloned the bre-1 gene and characterized the bre-1 mutant phenotype. bre-1 encodes a protein with significant homology to a GDP-mannose 4,6-dehydratase, which catalyzes the first step in the biosynthesis of GDP-fucose from GDP-mannose. Injection of GDP-fucose but not fucose into C. elegans intestinal cells rescues bre-1 mutant phenotypes. Thus, C. elegans lacks a functional fucose salvage pathway. Furthermore, we demonstrate that bre-1 mutant animals are defective in production of fucosylated glycolipids and that bre-1 mutant animals make quantitatively reduced levels of glycolipid receptors for Cry5B. We finally show that bre-1 mutant animals, although viable, show a lack of fucosylated N- and O-glycans, based on mass spectrometric evidence. Thus, C. elegans can survive with little fucose and can develop resistance to crystal toxin by loss of a monosaccharide biosynthetic pathway.

Mechanisms of ACTH(cAMP)-dependent transcription of adrenal steroid hydroxylases

The action of peptide hormones from the anterior pituitary regulates transcription of a large number of genes located in most, if not all, tissues. This action is mediated through regulation of steroid hormone production in the steroidogenic factories (adrenals, gonads). These steroid hormones are transported through the circulation to the peripheral tissues where they serve as ligands for the family of zinc-finger nuclear receptor transcription factors. The mechanisms by which peptide hormones regulate steroid hormone production include a chronic response mediated by elevated levels of cAMP resulting from the binding of peptide hormones to their cell surface receptors which enhances transcription of the genes encoding steroid hydroxylases required for steroid hormone biosynthesis. The action of ACTH in the adrenal cortex has been studied in greatest detail leading to identification of unique cAMP-response sequences (CRS) in the different bovine steroid hydroxylase genes. Most likely FSH and LH mediate steroid hydroxylase gene expression in the gonads via the same response elements. Unlike developmental/tissue-specific transcription of these genes which is regulated by a common transcription factor (SF-1), cAMP-dependent transcription of each steroid hydroxylase gene requires a different transcription factor.

Resistance to Bt Toxin in Caenorhabditis elegans from loss of Fucose

A mutation in the Caenorhabditis elegans bre-1 gene was isolated in a screen for Bacillus thuringiensis toxin-resistant (bre) mutants to the Cry5B crystal toxin made by B. thuringiensis. bre-1 mutant animals are different from the four other cloned bre mutants in that their level of resistance is noticeably lower. bre-1 animals also display a significantly reduced brood size at 25 °C. Here we cloned the bre-1 gene and characterized the bre-1 mutant phenotype. bre-1 encodes a protein with significant homology to a GDP-mannose 4,6-dehydratase, which catalyzes the first step in the biosynthesis of GDP-fucose from GDP-mannose. Injection of GDP-fucose but not fucose into C. elegans intestinal cells rescues bre-1 mutant phenotypes. Thus, C. elegans lacks a functional fucose salvage pathway. Furthermore, we demonstrate that bre-1 mutant animals are defective in production of fucosylated glycolipids and that bre-1 mutant animals make quantitatively reduced levels of glycolipid receptors for Cry5B. We finally show that bre-1 mutant animals, although viable, show a lack of fucosylated N- and O-glycans, based on mass spectrometric evidence. Thus, C. elegans can survive with little fucose and can develop resistance to crystal toxin by loss of a monosaccharide biosynthetic pathway.