Influence of ensiling time and inoculation on alteration of the starch-protein matrix in high-moisture corn.

The fates of hydrophobic zein proteins, which encapsulate corn starch to create vitreous endosperm, have not been investigated in high-moisture corn (HMC). To assess influences of ensiling time and inoculation on zein proteins in HMC, quadruplicate samples of 2 random corn hybrids (A and B), containing 25.7 and 29.3% moisture, were ground, inoculated with (I) or without 600,000 cfu/g of Lactobacillus buchneri 40788 (Lallemand Animal Nutrition, Milwaukee, WI), and ensiled for 0, 15, 30, 60, 120, and 240 d. Nutrient composition [crude protein (CP), starch, acid detergent fiber, and neutral detergent fiber], fermentation (pH, lactate, and acetate), and protein degradation markers (buffer-soluble CP, isopropanol-soluble CP, and NH(3)-N) were evaluated. At 0 and 240 d, α, γ, δ, and ß zein subunits were profiled using HPLC. Data were evaluated as a split-split plot using the PROC MIXED procedures of SAS. Ensiling time and inoculation decreased pH, and altered lactate and acetate contents of HMC. Lactate and acetate contents of A, AI, B, and BI at 240 d were 0.40, 0.32, 1.11, 0.73, and 0, 0.35, 0.30, and 0.87% of DM, respectively. Buffer-soluble CP in HMC increased from 1.5 to 2.0% of DM at 0 d to >4.0% of DM at 240 d. Inoculation had no effect on buffer-soluble CP but increased NH(3)-N content of HMC. Corn A contained more isopropanol-soluble CP than did corn B and peak areas for 6 α, and all γ and δ zein regions were greater for corn A. Ensiling (0 vs. 240 d) decreased all zein subunits with the exception of 2 α and 1 δ subunit. Ensiling decreased (42.2-73.2%) γ zeins, which are primarily responsible for cross-linking in the starch-protein matrix. Despite altering lactate and acetate contents, inoculation had no effect on degrading hydrophobic zein proteins in HMC. Data suggest that hydrophobic zein proteins in the starch-protein matrix of HMC are degraded by proteolytic activity over an extended ensiling time.

Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes.

1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200 microM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 microM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 microM BHT. 3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels. 4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6beta-hydroxylase activity. 5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.

Host shift by the burying beetle, Nicrophorus pustulatus, a parasitoid of snake eggs.

Recent work [Ecoscience (2000) vol. 7, 395-397] suggests that the burying beetle Nicrophorus pustulatus may have undergone a remarkable host shift, exploiting snake eggs rather than carrion as resources for breeding. We conducted behavioural and physiological experiments to examine the hypothesis of a host shift and to formulate hypotheses on its origin. Two congeners of N. pustulatus, Nicrophorus orbicollis and Nicrophorus defodiens did not respond to snake eggs with typical breeding behaviour. When N. pustulatus male-female pairs (n = 14) were presented with clutches of snake eggs, the number of offspring but not the mean size of offspring varied with snake egg mass, indicating effective regulation of brood size. When breeding on turtle eggs, N. pustulatus had a more variable response than when exploiting snake eggs, suggesting that turtle eggs are not a primary resource for breeding. Nicrophorus pustulatus presented with both snake eggs and a mouse carcass combined and exploited the two resources within the same nest (10 of 12 trials). Mouse carcasses and snake eggs were treated differently. Carcasses were moved, buried and stripped of hair in a manner characteristic of burying beetles, whereas snake eggs were not moved or buried. Females that discovered a mouse carcass also had a significantly greater juvenile hormone increase than did females discovering snake eggs. Some responses to the two resources, however, were similar. Female N. pustulatus oviposited rapidly in response to either a mouse carcass or snake eggs, and males elevated sex pheromone emission in response to either resource. The efficient use of snake eggs, the ability to regulate brood size and the different responses to snake eggs and carrion suggest that N. pustulatus is well adapted to exploiting snake eggs for breeding. The use of snake eggs by N. pustulatus has potential implications for conservation of oviparous reptiles.

Light is essential for degradation of ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit during sudden death syndrome development in soybean.

FUSARIUM SOLANI f. sp. GLYCINES (Fsg) has been reported to produce at least two phytotoxins. Cell-free FSG culture filtrates containing phytotoxins have been shown to develop foliar sudden death syndrome (SDS) in soybean. We have investigated the changes in protein profiles of diseased leaves caused by cell-free FSG culture filtrates prepared from FSG isolates. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) was conducted to investigate the protein profiles of diseased and healthy leaves. An approximately 55 kDa protein was found to be absent in diseased leaves. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometric analyses and a database search revealed that the missing protein is the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, which is involved in carbon assimilation and photorespiration. This result was confirmed by Western blot experiments. We have shown that light is essential for disappearance of the Rubisco large subunit initiated by cell-free FSG culture filtrates. The disappearance of the protein is fairly rapid and occurs within 24 h, presumably due to degradation. Cell-free, FSG culture-induced degradation of the Rubisco large subunit was accompanied by accumulation of reactive oxygen species under light conditions. Terminal deoxynucleotidyl transferase-mediated nick end labelling experiments suggested that programmed cell death was initiated in leaves of seedlings fed with cell-free FSG culture filtrates. These results suggest that, in the presence of light, FSG culture filtrates containing phytotoxins cause degradation of the Rubisco large subunit and accumulation of free radicals and, thereby, initiate programmed cell death leading to foliar SDS development in soybean.

Use of cultured precision-cut rat lung slices to study the in vitro induction of pulmonary cytochrome P450 forms.

1. The aim was to investigate the effects of some cytochrome P450 (CYP) enzyme inducers on CYP1A and CYP2B subfamily forms in cultured precision-cut rat lung slices. 2. Precision-cut lung slices were prepared from male Sprague-Dawley rats and cultured for 24 and/or 48 h in medium containing 0-20 micro g ml(-1) Aroclor 1254 (ARO), 0-50 micro M beta-naphthoflavone (BNF) and 0-50 micro M benzo(a)pyrene (BP). 3. Treatment with ARO, BNF and BP produced significant increases in lung slice whole homogenate 7-ethoxyresorufin O-deethylase activity. 4. Levels of CYP1A1 apoprotein were markedly increased in lung slice microsomes after treatment for 48 h with either 10 micro g ml(-1) ARO or 5 micro M BNF. In contrast, neither ARO nor BNF had any marked effect on levels of CYP2B1/2 apoprotein in 48-h cultured rat lung slice microsomes. 5. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan) was used to quantify lung slice CYP1A1 and CYP2B1/2 mRNA levels. Rat lung slice CYP1A1 mRNA levels were increased up to 8.3-fold after treatment for 24 h with 2 and 10 micro g ml(-1) ARO, 0.5 and 5 micro M BNF, and 20 micro M BP. In contrast, treatment with 10 micro g ml(-1) ARO produced only a small 1.6-fold increase in CYP2B1/2 mRNA levels. 6. Precision-cut lung slices are a useful model in vitro system for the assessment of the effects of chemicals on pulmonary CYP forms.

Studies on the induction of rat hepatic CYP1A, CYP2B, CYP3A and CYP4A subfamily form mRNAs in vivo and in vitro using precision-cut rat liver slices.

1. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan(R)) was used to examine the induction of some selected rat hepatic cyto-chrome P450 (CYP) forms in vivo and in vitro using cultured precision-cut liver slices. 2. TaqMan primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1, CYP2B1/2, CYP3A1, CYP3A2 and CYP4A1 mRNAs. 3. To characterize the responsiveness of the rat CYP mRNA TaqMan primers and probe sets, rats were treated in vivo with a single intraperitoneal dose of 500 mg kg(-1) Aroclor 1254 (ARO) and with four daily oral doses of either 50 mg kg(-1) day(-1) dexamethasone (DEX) or 75 mg kg(-1) day(-1) methylclofenapate (MCP). Treatment with ARO produced 22 600-, 5480-, 648-, 52-, 47- and 9-fold increases in levels of CYP1A1, CYP2B1, CYP2B1/2, CYP1A2, CYP3A1 and CYP3A2 mRNA, respectively. DEX treatment produced 97-, 24-, 8- and 4-fold increases, respectively, in CYP3A1, CYP2B1, CYP2B1/2 and CYP3A2 mRNA levels, and MCP produced 339-, 126- and 25-fold increases, respectively, in CYP4A1, CYP2B1 and CYP2B1/2 mRNA levels. All three CYP inducers also increased microsomal CYP content and produced corresponding increases in CYP1A, CYP2B, CYP3A and CYP4A form marker enzyme activities. 4. Rat liver slices were cultured for 6 and 24 h in medium containing 0.1 micro M insulin and 0.1 micro M DEX, and also for 24 h in medium containing only 0.1 micro M insulin (DEX-free medium). Liver slices were cultured in control medium or in medium containing either 10 micro M beta-naphthoflavone (BNF), 10 micro g ml(-1) ARO, 500 micro M sodium phenobarbitone (NaPB), 20 micro M pregnenolone-16alpha -carbonitrile (PCN), 50 micro M Wy-14,643 (WY) or 50 micro M MCP. 5. With the exception of the effect of BNF on CYP1A1 mRNA levels, the induction of all the CYP mRNAs studied was greater after 24- than after 6-h treatment. Generally, the magnitude of induction of CYP mRNA levels was greater after 24 h in liver slices cultured in DEX-free than in DEX-supplemented medium. 6. Treatment of liver slices with BNF and ARO for 24 h in DEX-free medium produced 21- and 35-fold increases, respectively, and 38- and 37-fold increases, respectively, in CYP1A1 and CYP1A2 mRNA levels. NaPB, PCN, WY and MCP did not increase either CYP1A1 or CYP1A2 mRNA levels. 7. After 24 h, levels of CYP2B1/2 mRNA were increased 18-, 20-, 9-, 16- and 13-fold by treatment with ARO, NaPB, PCN, WY and MCP, respectively. PCN also produced 56- and 4-fold increases, respectively, in CYP3A1 and CYP3A2 mRNA levels. 8. Treatment with WY and MCP for 24 h produced 437- and 186-fold increases, respectively, in levels of CYP4A1 mRNA. None of the other CYP inducers studied had any effect on CYP4A1 mRNA levels. 9. The results demonstrate the utility of cultured precision-cut liver slices as an in vitro model system to evaluate the effects of xenobiotics on rat CYP1A, CYP2B, CYP3A and CYP4A form mRNA levels.

A mouse model for medulloblastoma and basal cell nevus syndrome.

Medulloblastoma (MB), a tumor of the cerebellum, is the most frequent type of malignant childhood brain tumor. Multiple genes are causally involved in medulloblastoma including PATCHED1 (PTCH). The Patchedl (Ptc1) protein is a receptor for Sonic hedgehog (Shh), a secreted protein ligand. Shh is involved in many signaling processes that control cell fate and growth, among which is its emission from Purkinje cells in the developing cerebellum. Purkinje cell-derived Shh stimulates mitosis of the granule cell precursors that may be the cell type of origin in medulloblastoma. Ptc1 limits the effects of the Shh signal, so mutations in PTCH may lead to persistent granule cell precursors susceptible to further genetic or environmental events that cause medulloblastoma. Mice heterozygous for patched (ptc1) mutations, like heterozygous PTCH humans, have a high rate of medulloblastoma as well as other tumors. We discuss features of the mouse model and how it is contributing to understanding the process of brain tumorigenesis.

Jelly belly: a Drosophila LDL receptor repeat-containing signal required for mesoderm migration and differentiation.

Inductive interactions subdivide the Drosophila mesoderm into visceral, somatic, and heart muscle precursors. The muscle precursors form organs by executing tissue-specific migrations and cell fusions. We identified a novel gene, jelly belly (jeb), which is required for visceral mesoderm development. jeb encodes a secreted protein that contains an LDL receptor repeat. In jeb mutants, visceral mesoderm precursors form, but they fail to migrate or differentiate normally; no visceral muscles develop. Jeb protein is produced in somatic muscle precursors and taken up by visceral muscle precursors. jeb reveals a signaling process in which somatic muscle precursors support the proper migration and differentiation of visceral muscle cells. Later in embryogenesis, jeb is transcribed in neurons and Jeb protein is found in axons.

Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases.

Steroid hormones have rapid nongenomic effects on cell-signaling pathways, but the receptor mechanisms responsible for this are not understood. We have identified a specific polyproline motif in the amino-terminal domain of conventional progesterone receptor (PR) that mediates direct progestin-dependent interaction of PR with SH3 domains of various cytoplasmic signaling molecules, including c-Src tyrosine kinases. Through this interaction, PR is a potent activator of Src kinases working by an SH3 domain displacement mechanism. By mutagenesis, we also show that rapid progestin-induced activation of Src and downstream MAP kinase in mammalian cells is dependent on PR-SH3 domain interaction, but not on the transcriptional activity of PR. Preliminary evidence for the biological significance of this PR signaling pathway through regulatory SH3 domains was shown with respect to an influence on progestin-induced growth arrest of breast epithelial cells and induction of Xenopus oocyte maturation.

Patterns of gene expression during Drosophila mesoderm development.

The transcription factor Twist initiates Drosophila mesoderm development, resulting in the formation of heart, somatic muscle, and other cell types. Using a Drosophila embryo sorter, we isolated enough homozygous twist mutant embryos to perform DNA microarray experiments. Transcription profiles of twist loss-of-function embryos, embryos with ubiquitous twist expression, and wild-type embryos were compared at different developmental stages. The results implicate hundreds of genes, many with vertebrate homologs, in stage-specific processes in mesoderm development. One such gene, gleeful, related to the vertebrate Gli genes, is essential for somatic muscle development and sufficient to cause neural cells to express a muscle marker.

Vertebrate proteins related to Drosophila Naked Cuticle bind Dishevelled and antagonize Wnt signaling.

Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In the fruit fly Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Here we describe two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals.

The HMG-domain protein BAP111 is important for the function of the BRM chromatin-remodeling complex in vivo.

The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a SWI/SNF-like chromatin-remodeling complex. A key question about chromatin-remodeling complexes is how they interact with DNA, particularly in the large genomes of higher eukaryotes. Here, we report the characterization of BAP111, a BRM-associated protein that contains a high mobility group (HMG) domain predicted to bind distorted or bent DNA. The presence of an HMG domain in BAP111 suggests that it may modulate interactions between the BRM complex and chromatin. BAP111 is an abundant nuclear protein that is present in all cells throughout development. By using gel filtration chromatography and immunoprecipitation assays, we found that the majority of BAP111 protein in embryos is associated with the BRM complex. Furthermore, heterozygosity for BAP111 enhanced the phenotypes resulting from a partial loss of brm function. These data demonstrate that the BAP111 subunit is important for BRM complex function in vivo.

The developmental biology of brain tumors.

Tumors of the central nervous system (CNS) can be devastating because they often affect children, are difficult to treat, and frequently cause mental impairment or death. New insights into the causes and potential treatment of CNS tumors have come from discovering connections with genes that control cell growth, differentiation, and death during normal development. Links between tumorigenesis and normal development are illustrated by three common CNS tumors: retinoblastoma, glioblastoma, and medulloblastoma. For example, the retinoblastoma (Rb) tumor suppressor protein is crucial for control of normal neuronal differentiation and apoptosis. Excessive activity of the epidermal growth factor receptor and loss of the phosphatase PTEN are associated with glioblastoma, and both genes are required for normal growth and development. The membrane protein Patched1 (Ptc1), which controls cell fate in many tissues, regulates cell growth in the cerebellum, and reduced Ptc1 function contributes to medulloblastoma. Just as elucidating the mechanisms that control normal development can lead to the identification of new cancer-related genes and signaling pathways, studies of tumor biology can increase our understanding of normal development. Learning that Ptc1 is a medulloblastoma tumor suppressor led directly to the identification of the Ptc1 ligand, Sonic hedgehog, as a powerful mitogen for cerebellar granule cell precursors. Much remains to be learned about the genetic events that lead to brain tumors and how each event regulates cell cycle progression, apoptosis, and differentiation. The prospects for beneficial work at the boundary between oncology and developmental biology are great.

Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events.

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.

Naked cuticle targets dishevelled to antagonize Wnt signal transduction.

In Drosophila embryos the protein Naked cuticle (Nkd) limits the effects of the Wnt signal Wingless (Wg) during early segmentation. nkd loss of function results in segment polarity defects and embryonic death, but how nkd affects Wnt signaling is unknown. Using ectopic expression, we find that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg signal, and the role of Wg is to relieve this inhibition. Our double-mutant analysis shows that, in contrast to Zw3, Nkd acts when the Wg pathway is active to restrain signal transduction. Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. Our results suggest that Nkd acts directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/beta-catenin and activate downstream genes.

Automated sorting of live transgenic embryos.

The vast selection of Drosophila mutants is an extraordinary resource for exploring molecular events underlying development and disease. We have designed and constructed an instrument that automatically separates Drosophila embryos of one genotype from a larger population of embryos, based on a fluorescent protein marker. This instrument can also sort embryos from other species, such as Caenorhabditis elegans. The machine sorts 15 living Drosophila embryos per second with more than 99% accuracy. Sorting living embryos will solve longstanding problems, including (1) the need for large quantities of RNA from homozygous mutant embryos to use in DNA microarray or gene-chip experiments, (2) the need for large amounts of protein extract from homozygous mutant embryos for biochemical studies, for example to determine whether a multiprotein complex forms or localizes correctly in vivo when one component is missing, and (3) the need for rapid genetic screening for gene expression changes in living embryos using a fluorescent protein reporter.

Interleukin-10 is an unequivocal Th2 parameter in the rat, whereas interleukin-4 is not.

Exposure of Wistar rats to the immunotoxic compounds hexachlorobenzene (HCB), bis(tri-n-butyltin)oxide, and benzo(a)pyrene was previously found to affect mRNA expression of interleukin (IL)-2, IL-2R alpha-chain, and interferon (IFN)-gamma, the prototypic Th1 cytokine. In contrast, the mRNA expression of IL-4, the prototypic Th2 cytokine, was unaffected. This latter finding suggested that the IL-4 mRNA expression may not be an unequivocal parameter for Th2 responses in the rat. In order to obtain such a parameter the present study was performed, consisting of two types of experiments. Expression and production of IL-4 as well as IL-10, a second Th2 cytokine, were measured. First, Lewis (Th1 prone) and Brown Norway (BN; Th2 prone) rats were exposed to HCB. Exposure was previously found to increase the serum immunoglobulin (Ig)E levels, an IL-4-dependent response, in BN but not Lewis rats, and in Lewis rats to aggravate experimental allergic encephalomyelitis (EAE), severity being inversely related to IL-10 levels. Secondly, BN rats were infected with Trichinella spiralis, an infection previously found to induce IL-4 production. HCB exposure did not affect IL-4 mRNA expression in either strain, while IL-4 production was decreased in Lewis and unaffected in BN rats. In Lewis rats both the mRNA expression and the production of IL-10 were decreased. The T. spiralis infection induced IL-4 and IL-10 mRNA expression, as well as IL-10 production. In contrast, the IL-4 production was strongly reduced. Thus, both the IL-10 mRNA expression and production correlated with the EAE development and T. spiralis infection. In HCB exposed Lewis rats and T. spiralis infected BN rats the IL-4 mRNA expression correlated with IgE levels and T. spiralis infection, respectively, whereas the IL-4 production lacked correlation in all cases. Collectively, these results suggest that IL-10 is an unequivocal Th2 parameter in the rat, whereas IL-4 is not.

A peptide model system for processive phosphorylation by Src family kinases.

The Src homology 2 (SH2) and Src homology 3 (SH3) domains of Src family kinases are involved in substrate recognition in vivo. Many cellular substrates of Src kinases contain a large number of potential phosphorylation sites, and the SH2 and SH3 domains of Src are known to be required for phosphorylation of these substrates. In principle, Src could phosphorylate these substrates by either a processive mechanism, in which the enzyme remains bound to the peptide substrate during multiple phosphorylation events, or a nonprocessive (distributive) mechanism, where each phosphorylation requires a separate binding interaction between enzyme and substrate. Here we use a synthetic peptide system to demonstrate that Hck, a Src family kinase, can phosphorylate substrates containing an SH2 domain ligand by a processive mechanism. Hck catalyzes the phosphorylation of these sites in a defined order. Furthermore, we show that addition of an SH3 domain to a peptide can enhance its phosphorylation both by activating Hck and by increasing the affinity of the substrate. On the basis of our observations on the role of the SH2 and SH3 domains in substrate recognition, we present a model for substrate targeting in vivo.

In vivo functions of the patched protein: requirement of the C terminus for target gene inactivation but not Hedgehog sequestration.

The membrane protein Patched (Ptc) is a key regulator of Hedgehog (Hh) signaling in development and is mutated in human tumors. Ptc opposes Hh-induced gene transcription and sequesters Hh protein. To dissect these functions, we tested partially deleted forms of Ptc in Drosophila. Deletion of either half of Ptc abolishes all function while coexpression of the halves restores nearly full activity. Deletion of the final 156 residues of Ptc permits Hh sequestration but abolishes inhibition of Hh targets. This deletion has dominant-negative activity, promoting target gene activation in a ligand-independent manner. We observe little or no association of full-length or partially deleted Ptc with the membrane protein Smoothened in Drosophila cultured cells.

Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine.

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.