Immunohistochemical localization of FAP-1, an inhibitor of Fas-mediated apoptosis, in normal and neoplastic human tissues

Fas, a death receptor, is widely expressed in human tissue, but its expression, although a prerequisite for the induction of apoptosis, does not predict its biological function. To understand the mechanisms of Fas resistance in human tissues in vivo, we performed immunohistochemistry using an antibody against Fas-associated phosphatase-1 (FAP-1), which interacts with the cytosolic domain of Fas and inhibits Fas-mediated apoptosis. In normal human tissues, FAP-1 immunostaining was easily detected, for example, in renal tubules, skeletal muscle, myocardiocytes, pituitary gland, parathyroid gland, pancreatic islets, hepatocytes, testicular germ cells, prostatic glands, neurons, epithelium of fallopian tube, endometrial glands, trophoblasts, bronchial epithelial cells, and some types of gastrointestinal epithelial cells. In 123 (78%) of 158 cancers of various origins, including breast carcinomas, stomach carcinomas, colon carcinomas, lung carcinomas and several types of sarcomas, variable intensities of FAP-1 expression were evident. Taken together, these findings demonstrated that FAP-1 is widely expressed in normal human tissues and partly overlapped with Fas expression described in earlier reports, suggesting that FAP-1 may have an important role in the regulation of apoptosis in vivo. In addition, FAP-1 expression in cancers suggests that many cancers may be resistant to Fas-mediated apoptosis through the action of FAP-1 in vivo.

Paracrine regulation of ovarian cancer by endothelin

Previous studies have demonstrated that endothelin (ET) isoforms (ET-1, ET-2 and ET-3) can act in an autocrine manner in ovarian cancer while in breast cancer their role has been proposed to be that of a paracrine mitogen. To explore the possibility that endothelin isoforms might function not only as autocrine regulators but also as paracrine mitogens in ovarian cancers, we investigated their effects on the growth of ovarian fibroblasts derived from ovarian carcinomas, the interaction between ovarian carcinoma and fibroblast cells and the location of the isoform expression in primary ovarian tumours. ET-1, ET-2 and ET-3 stimulated the growth of three ovarian fibroblast cell lines at concentrations ranging from 10(-12) M to 10(-7) M. Inhibition of 125I-ET binding by the ETA receptor antagonist BQ123 and the ETB receptor antagonist BQ788 suggested the presence of both types of ET receptors in fibroblast cells. In the absence of ET-1, neither BQ 123 nor BQ 788 inhibited growth. However, both antagonists inhibited ET-1 stimulated growth suggesting the involvement of both receptor types in ET-1 growth regulation. In contrast to carcinoma cells which secrete measurable levels of ET-1, fibroblast cell lines did not secrete detectable protein. Co-culture experiments (using porous membrane insert wells) of fibroblasts with carcinoma cells demonstrated that growth of both populations of cells was increased compared with either grown in isolation. In this system, growth of the fibroblast cell line was partially inhibited by both BQ123 and BQ788, whilst growth of the PE014 carcinoma cell line was inhibited by only BQ123. RT-PCR measurements detected the presence of the ETA receptor subtype in 10/10 primary ovarian cancers but the presence of ETB receptor in only 6/10 cancers. Using specific antibodies, ET-1 was found in 11/15, ET-2 in 5 of 7 and ET-3 in 5/7 primary ovarian cancers predominantly in the epithelial cells but with some stromal expression. These data indicate that the ET isoforms may stimulate growth of the fibroblast population within an ovarian cancer in addition to stimulating the epithelial cells and since the ETs are expressed in the majority of ovarian cancers, this paracrine effect may contribute to the overall growth of the tumour.

Pharmacological characterization of receptor-mediated Ca2+ entry in endothelin-1-induced catecholamine release from cultured bovine adrenal chromaffin cells

To clarify the mechanism for the endothelin-1 (ET-1)-induced release of catecholamines from the adrenal gland, we examined the effects of removal of extracellular Ca2+, blockers of L-, N-, P- and Q-types of voltage-operated Ca2+ channels (VOCC) such as nifedipine (L-type), omega-conotoxin GVIA (N-type), omega-agatoxin IVA (P-type) and omega-conotoxin MVIIC (Q-type) and blockers of voltage-independent Ca2+ entry channel such as SKF 96365 and LOE 908 on release of catecholamines, the cytosolic free Ca2+ concentration ([Ca2+]i), and 45Ca2+ uptake in cultured bovine adrenal chromaffin cells. ET-1 but not ET-3 induced increases in release of catecholamines, [Ca2+]i, and 45Ca2+ uptake. The responses to ET-1 were abolished by the antagonist for ET(A) receptors, BQ-123, but not by the antagonist for ET(B) receptors, BQ-788, and they were abolished by removal of extracellular Ca2+. The increases were only partially inhibited (to about 65% of control) by nifedipine but unaffected by any of the omega-toxins. The nifedipine-resistant increase was inhibited by SKF 96365 (to about 40%) and abolished by LOE 908 alone. These results indicate that ET-1 augments the release of catecholamines from adrenal chromaffin cells through ET(A) receptors, by activating two types of Ca2+ entry channels in addition to L-type VOCC: one (nonselective cation channel-1; NSCC-1) is sensitive to LOE 908 but resistant to SKF 96365, whereas the other (NSCC-2) is sensitive to both LOE 908 and SK&F 96365.

Metabolism-based polycyclic aromatic acetylene inhibition of CYP1B1 in 10T1/2 cells potentiates aryl hydrocarbon receptor activity

We have used polycyclic aromatic hydrocarbon (PAH) alkyne metabolism-based inhibitors to test whether CYP1B1 metabolism is linked to aryl hydrocarbon receptor (AhR) activation in mouse embryo fibroblasts (MEF). 1-ethynylpyrene (1EP) selectively inactivated CYP1B1 dimethylbenzanthracene (DMBA) metabolism in C3H10T1/2 MEFs; whereas 1-(1-propynyl)pyrene (1PP) preferentially inhibited CYP1A1 activity in Hepa-1c1c7 mouse hepatoma cells (Hepa). In each cell type >90% inhibition of DMBA metabolism after 1 h treatment with each inhibitor (0.1 microM) was progressively reversed and then increased to levels seen with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induction (fourfold stimulation). It was found that 0.1 microM 1EP and 1PP maximally induce CYP1B1 and CYP1A1 mRNA levels in 10T1/2 and Hepa cells, respectively, after 6 h. 1-Ethylpyrene (EtP), which lacks the activatable acetylene moiety, was far less effective as an inhibitor and as an inducer. AhR activation is essential for 1EP induction as evidenced by the use of AhR antagonists and AhR-deficient MEFs and absence of induction following inhibition of DMBA metabolism with carbon monoxide (CO). Inhibition of CYP1B1 was linked to enhanced AhR activation even at early stages prior to significant ligand depletion. 1EP and EtP were similarly effective in stimulating AhR nuclear translocation, though 5-10 times slower compared with TCDD, and produced no significant down-regulation of the AhR. TCDD activated AhR/Arnt complex formation with an oligonucleotide xenobiotic response element far more extensively than 1EP or EtP, even at concentrations of 1EP that increased CYP1B1 mRNA to similar levels. CO did not influence these responses to EtP, event hough CO treatment potentiated EtP induction of CYP1B1 mRNA. These differences suggest a fundamental difference between PAH/AhR and TCDD/AhR complexes where CYP1B1 metabolic activity regulates the potency, rather than the formation of the AhR/Arnt complex. Copyright 1999 Academic Press.

Endothelin-1 activates p38 mitogen-activated protein kinase via endothelin-A receptor in rat myocardial cells

In myocardial cells (MCs), endothelin-1 (ET-1) exerts various effects such as hypertrophy, and causes cellular injury. Long-term treatment with an endothelin-A (ET(A)) receptor antagonist improves the survival of rats with heart failure, suggesting that myocardial endothelin system contributes to the progression of heart failure. p38 mitogen-activated kinase (MAPK) is a member of the MAPK family and activated by several forms of environmental stresses. We show here the effect of ET-1 on p38 MAPK activation and the role of ET-1-activated p38 MAPK on morphological changes in MCs. ET-1-stimulated p38 MAPK phosphorylation was detectable within 2 min and maximal at 5 min and was concentration dependent. The maximum effect was obtained at 10 nM. An ET(A) receptor antagonist, BQ-123, but not an endothelin-B receptor antagonist, BQ-788, inhibited these reactions. A p38 MAPK inhibitor, SB203580, failed to inhibit the morphological changes associated with ET-1-induced myocardial cell hypertrophy. These results indicate that p38 MAPK is activated by ET-1 but does not contribute to the development of ET-1-induced myocardial cell hypertrophy.

Endothelin receptors and calcium translocation pathways in human airways

Tension and phosphatidyl inositol (PI) turnover experiments were conducted to investigate the receptors and signal transduction pathways responsible for contractions elicited by endothelin (ET) ligands in human bronchus. Nicardipine (1 microM), the L-type calcium channel inhibitor, or incubation in Ca2+-free medium, produced marked inhibition of contractions to the ET(B) receptor-selective agonist, sarafotoxin S6c, and especially those induced by KCl. In contrast, Ca2+-free medium was without appreciable effect against contraction produced by endothelin-1 (ET-1), the non-selective ET(A) and ET(B) receptor agonist. In Ca2+-free medium, ryanodine (10 microM), which inhibits intracellular calcium mobilization, reduced sarafotoxin S6c- and ET-1-induced responses, but was without effect on responses to KCl. Similarly, nickel chloride (Ni2+; 1 mM) caused marked inhibition of contractions induced by sarafotoxin S6c or ET-1, but had no significant effect on KCI concentration-response curves. The mixed ET(A)/ET(B) receptor antagonist SB 209670 (3 microM) inhibited responses to sarafotoxin S6c and ET-1 such that concentration-response curves were shifted rightward, at the 30% maximum response level, by 10.0- and 3.8-fold, respectively, whereas BQ-123 (3 microM), the ET(A) receptor antagonist, was without effect on response induced by either agonist. ET-1 (1 nM-0.3 microM) caused a concentration-dependent stimulation of PI turnover, whereas sarafotoxin S6c (0.3 nM-0.1 microM) induced only small and variable increases, except at the highest concentration. The increase in PI turnover evoked by ET-1 was inhibited by SB 209670 (3 microM), and also by BQ-123 (3 microM). This is consistent with linkage of ET(A) receptors to activation of inositol phosphate generation in human bronchial smooth muscle cells. Collectively, the data suggest that differences exist in the relative contributions of intracellular and extracellular Ca2+ mobilization mechanisms elicited by ET(A) and ET(B) receptor activation. Thus, sarafotoxin S6c-induced, ET(B) receptor-mediated contraction in human bronchial smooth muscle appears to be dependent, in part, upon extracellular Ca2+, although a significant component of the response was also mediated by intracellular Ca2+ release, including from ryanodine-sensitive stores. ET(A) receptor-mediated contraction of human airway smooth muscle was activated largely via the release of intracellular Ca2+.

Carbon-11 epidepride: a suitable radioligand for PET investigation of striatal and extrastriatal dopamine D2 receptors

Epidepride [(S)-(-)-N-([1-ethyl-2-pyrrolidinyl]methyl)-5-iodo-2,3-dimethoxybenza mide] binds with a picomolar affinity (Ki = 24 pM) to the dopamine D2 receptor. Iodine-123-labeled epidepride has been used previously to study striatal and extrastriatal dopamine D2 receptors with single photon emission computed tomography (SPECT). Our aim was to label epidepride with carbon-11 for comparative quantitative studies between positron emission tomography (PET) and SPECT. Epidepride was synthesized from its bromo-analogue FLB 457 via the corresponding trimethyl-tin derivative. In an alternative synthetic pathway, the corresponding substituted benzoic acid was reacted with the optically pure aminomethylpyrrolidine-derivative. Demethylation of epidepride gave the desmethyl-derivative, which was reacted with [11C]methyl triflate. Total radiochemical yield was 40-50% within a total synthesis time of 30 min. The specific radioactivity at the end of synthesis was 37-111 GBq/micromol (1,000-3,000 Ci/mmol). Human postmortem whole-hemisphere autoradiography demonstrated dense binding in the caudate putamen, and also in extrastriatal areas such as the thalamus and the neocortex. The binding was inhibited by unlabeled raclopride. PET studies in a cynomolgus monkey demonstrated high uptake in the striatum and in several extrastriatal regions. At 90 min after injection, uptake in the striatum, thalamus and neocortex was about 11, 4, and 2 times higher than in the cerebellum, respectively. Pretreatment experiment with unlabeled raclopride (1 mg/kg) inhibited 50-70% of [11C]epidepride binding. The fraction of unchanged [11C]epidepride in monkey plasma determined by a gradient high performance liquid chromatography (HPLC) method was about 30% of the total radioactivity at 30 min after injection of [11C]epidepride. The availability of [11C]epidepride allows the PET-verification of the data obtained from quantitation studies with SPECT.

p27 expression as a prognostic factor of breast cancer in Taiwan

p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. It binds to a variety of cyclin/CDK complexes, inhibits kinase activity, and blocks the cell cycle. Absent or reduced p27 expression has been shown to be a significant predictor of poor survival in breast, colorectal, prostate, non-small cell lung and esophagus carcinomas. An immunohistochemical assay was performed on 169 patients with primary breast cancers to evaluate the biologic significance of p27 expression. Decreased p27 expression was significantly associated with high grade (P = 0.00025), negative estrogen receptor (P = 0.00004), and negative progesterone receptor (P = 0.0038) breast cancers. Univariate analysis reveals that p27 expression inversely correlated significantly with overall survival (P = 0.0001). By multivariate analysis, p27 predicted the overall survival independently (P = 0.0096). Our study indicates that p27 expression is an independent prognostic marker of breast cancer in Taiwan.

Developmental conversion of inotropism by endothelin I and angiotensin II from positive to negative in mice

Inotropic effects on isolated neonatal and adult mouse myocardium of endothelin I and angiotensin II were examined. Endothelin I produced a sustained positive inotropic response in the neonate but a sustained negative response in the adult. Both were concentration-dependent and were inhibited by the endothelin ETA receptor antagonist, BQ-123 (Cyclo(D-a-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)). Angiotensin II produced a sustained positive inotropic response in the neonate while a sustained negative response in the adult. Both were concentration-dependent and were inhibited by the angiotensin AT1 receptor antagonist, YM358 (2,7-diethyl-5-((2'-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl-5H-pyrazolo(1,5-b)(1,2,4)tria zole potassium salt monohydrate). These results indicate that inotropic responses of the mouse heart to cardioactive peptides are unique among experimental animal species and may be reversed during development.

The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins

Cleavage and polyadenylation specificity factor (CPSF), a key component of the mammalian RNA 3'-end processing machinery, consists of four subunits of 160, 100, 73, and 30 kD. Here we report the isolation and characterization of a cDNA encoding the 30-kD polypeptide. Antibodies raised against this protein inhibit cleavage and polyadenylation and coimmunoprecipitate the other CPSF subunits. The protein sequence contains five C3H-zinc-finger repeats and a putative RNA-binding zinc knuckle motif at the carboxyl terminus. Consistent with this observation, the in vitro translated 30-kD protein binds RNA polymers with a distinct preference for poly(U). In addition, an essential S. cerevisiae gene, YTH1, was cloned which is 40% identical to CPSF 30K at the protein level. Extracts prepared from a conditional yth1 mutant have normal cleavage activity, but fail to polyadenylate the upstream cleavage product. Efficient polyadenylation activity can be restored by the addition of purified polyadenylation factor I (PF I). We demonstrate that Yth1p is a component of PF I that interacts in vivo and in vitro with Fip1p, a known PF I subunit.

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiac sarcolemmal vesicles, MgATP stimulates Na+/Ca2+ exchange with the following characteristics: 1) increases 10-fold the apparent affinity for cytosolic Ca2+; 2) a Michaelis constant for ATP of approximately 500 microM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 microM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5'-O-(3-thiotriphosphate), without the need for vanadate, but not by beta,gamma-methyleneadenosine 5'-triphosphate; and 6) not affected by unspecific protein alkaline phosphatase but abolished by a phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC effect is counteracted by phosphatidylinositol. In addition, in the absence of ATP, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) was able to stimulate the exchanger activity in vesicles pretreated with PI-PLC. This MgATP stimulation is not related to phosphorylation of the carrier, whereas phosphorylation appeared in the phosphoinositides, mainly PIP2, that coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP and no Ca2+ show a marked synthesis of L-alpha-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 microM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2 increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+ exchanger through a fast phosphorylation chain: a Ca(2+)-independent PIP formation followed by a Ca(2+)-dependent synthesis of PIP2.

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton

p70 S6 kinase (p70(S6k)) is a mitogen-activated protein kinase that plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1. Here, we report the identification of neurabin (neural tissue-specific F-actin binding protein), a neuronally enriched protein of 1,095 amino acids that contains a PDZ domain and binds p70(S6k). We demonstrate the neurabin-p70(S6k) interaction by yeast two-hybrid analysis and biochemical techniques. p70(S6k) and neurabin coimmunoprecipitate from transfected HEK293 cells. Site-directed mutagenesis of neurabin implicates its PDZ domain in the interaction with p70(S6k), and deletion of the carboxyl-terminal five amino acids of p70(S6k) abrogates the interaction. Cotransfection of neurabin in HEK293 cells activates p70(S6k) kinase activity. The mRNA of neurabin and p70(S6k) show striking colocalization in brain sections by in situ hybridization. Subcellular fractionation of rat brain demonstrates that neurabin and p70(S6k) both localize to the soluble fraction of synaptosomes. By way of its PDZ domain, the neuronal-specific neurabin may target p70(S6k) to nerve terminals.

The leukemic protein core binding factor beta (CBFbeta)-smooth-muscle myosin heavy chain sequesters CBFalpha2 into cytoskeletal filaments and aggregates

The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor beta (CBFbeta) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFbeta-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFalpha1 to 3) and a non-DNA binding subunit (CBFbeta). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBFbeta-SMMHC, CBFbeta, and CBFalpha2 (also known as AML1 or PEBP2alphaB). When expressed individually, CBFalpha2 was located in the nuclei of transfected cells, whereas CBFbeta was distributed throughout the cell. On the other hand, CBFbeta-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFalpha2 was able to drive localization of CBFbeta into the nucleus in a dose-dependent manner. In, CBFalpha2 colocalized with CBFbeta-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFalpha-interacting domain within CBFbeta-SMMHC abolished this CBFalpha2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFbeta-SMMHC when coexpressed with CBFalpha2. CBFalpha2 sequestration by CBFbeta-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFbeta-SMMHC plays a dominant negative role by sequestering CBFalpha2 into cytoskeletal filaments and aggregates, thereby disrupting CBFalpha2-mediated regulation of gene expression.

Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation

Differentiating cells undergo programmed alterations in their patterns of gene expression, which are often regulated by structural changes in chromatin. Here we demonstrate that T cell differentiation results in long-range changes in the chromatin structure of effector cytokine genes, which persist in resting Th1 and Th2 cells in the absence of further stimulation. Differentiation of naive T helper cells into mature Th2 cells is associated with chromatin remodeling of the IL-4 and IL-13 genes, whereas differentiation into Th1 cells evokes remodeling of the IFNgamma but not IL-4 or IL-13 genes. IL-4 locus remodeling is accompanied by demethylation and requires both antigen stimulation and STAT6 activation. We propose that chromatin remodeling of cytokine gene loci is functionally associated with productive T cell differentiation and may explain the coordinate regulation of Th2 cytokine genes.

Neural activity in the primate prefrontal cortex during associative learning [see comments]

The prefrontal (PF) cortex has been implicated in the remarkable ability of primates to form and rearrange arbitrary associations rapidly. This ability was studied in two monkeys, using a task that required them to learn to make specific saccades in response to particular cues and then repeatedly reverse these responses. We found that the activity of individual PF neurons represented both the cues and the associated responses, perhaps providing a neural substrate for their association. Furthermore, during learning, neural activity conveyed the direction of the animals' impending responses progressively earlier within each successive trial. The final level of activity just before the response, however, was unaffected by learning. These results suggest a role for the PF cortex in learning arbitrary cue-response associations, an ability critical for complex behavior.

Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRalpha3-RET receptor complex

The glial cell line-derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRalpha1-GFRalpha4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRalpha3-RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRalpha1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRalpha1-RET) and NTN (GFRalpha2-RET), Artemin has a preferred receptor (GFRalpha3-RET) but that alternative receptor interactions also occur.

Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.

Role of the lateral mammillary nucleus in the rat head direction circuit: a combined single unit recording and lesion study [published erratum appears in Neuron 1999 Jan;22(1):199]

We recorded head direction (HD) cells from the lateral mammillary nucleus (LMN) and anterior thalamus (ATN) of freely behaving rats and also made bilateral lesions of LMN while recording HD cells from ATN. We discovered that the tuning functions of LMN HD cells become narrower during contraversive head turns, but not ipsiversive head turns, compared to when the head is not turning. This narrowing effect does not occur for ATN HD cells. We also found that the HD signal in LMN leads that in ATN by about 15-20 ms. When LMN was lesioned bilaterally, HD cells in ATN immediately lost their directional firing properties and never recovered them. Based on these findings, we argue that LMN may be an essential component of an attractor-integrator network that participates in generating the HD signal.

Inducible nonlymphoid expression of Fas ligand is responsible for superantigen-induced peripheral deletion of T cells

Fas (CD95) and Fas ligand (FasL) play major roles in staphylococcal enterotoxin B (SEB)-induced peripheral deletion of Vbeta8+ T cells. We found that peripheral deletion was defective in radiation chimeras with non-functional tissue FasL, regardless of the FasL status of the bone marrow-derived cells. SEB induced a dramatic upregulation of FasL expression and function in nonlymphoid cells of liver and small intestine. This effect was resistant to inhibition by cyclosporin A, which also failed to inhibit peripheral deletion. In SCID animals nonlymphoid tissues did not express FasL in response to SEB unless transplanted lymphocytes were present. Thus, some immune responses induce FasL in nonlymphoid tissues, which in turn kills activated lymphocytes, leading to peripheral T cell deletion.

Regulation of PAK activation and the T cell cytoskeleton by the linker protein SLP-76

Tyrosine phosphorylation of linker proteins enables the T cell antigen receptor (TCR)-associated protein tyrosine kinases to phosphorylate and regulate effector molecules that generate second messengers. We demonstrate here that the SLP-76 linker protein interacts with both nck, an adaptor protein, and Vav, a guanine nucleotide exchange factor for Rho-family GTPases. The assembly of this tri-molecular complex permits the activated Rho-family GTPases to regulate target effectors that interact through nck. In turn, assembly of this complex mediates the enzymatic activation of the p21-activated protein kinase 1 and facilitates actin polymerization. Hence, phosphorylation of linker proteins not only bridges the TCR-associated PTK, ZAP-70, with downstream effector proteins, but also provides a scaffold to integrate distinct signaling complexes to regulate T cell function.

An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons

Olfactory sensory neurons expressing a given odorant receptor project to two topographically fixed glomeruli in the olfactory bulb. We have examined the contribution of different cell types in the olfactory bulb to the establishment of this topographic map. Mice with a homozygous deficiency in Tbr-1 lack most projection neurons, whereas mice with a homozygous deficiency in Dlx-1 and Dlx-2 lack most GABAergic interneurons. Mice bearing a P2-IRES-tau-lacZ allele and deficient in either Tbr-1 or Dlx-1/Dlx-2 reveal the convergence of axons to one medial and one lateral site at positions analogous to those observed in wild-type mice. These observations suggest that the establishment of a topographic map is not dependent upon cues provided by, or synapse formation with, the major neuronal cell types in the olfactory bulb.

Regulation of cocaine reward by CREB

Cocaine regulates the transcription factor CREB (adenosine 3', 5'-monophosphate response element binding protein) in rat nucleus accumbens, a brain region that is important for addiction. Overexpression of CREB in this region decreases the rewarding effects of cocaine and makes low doses of the drug aversive. Conversely, overexpression of a dominant-negative mutant CREB increases the rewarding effects of cocaine. Altered transcription of dynorphin likely contributes to these effects: Its expression is increased by overexpression of CREB and decreased by overexpression of mutant CREB. Moreover, blockade of kappa opioid receptors (on which dynorphin acts) antagonizes the negative effect of CREB on cocaine reward. These results identify an intracellular cascade-culminating in gene expression-through which exposure to cocaine modifies subsequent responsiveness to the drug.

Combinatorial signaling codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm

Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute (l'sc)-expressing cells. Using both genetic loss-of-function and targeted ectopic expression approaches, we demonstrate here that individual progenitors are specified by the sequential deployment of unique combinations of intercellular signals. Initially, the intersection between the Wingless (Wg) and Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on the adjacent mesoderm in the form of a L'sc precluster. All mesodermal cells within this precluster are competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases (RTKs), the Drosophila epidermal growth factor receptor (DER) and the Heartless (Htl) fibroblast growth factor receptor. By monitoring the expression of the diphosphorylated form of mitogen-associated protein kinase (MAPK), we found that these RTKs are activated in small clusters of cells within the original competence domain. Each cluster represents an equivalence group because all members initially resemble progenitors in theirr expression of both L'sc and mesodermal identity genes. Thus, localized RTK activity induces the formation of mesodermal equivalence groups. The RTKs remain active in the single progenitor that emerges from each cluster under the subsequent inhibitory influence of the neurogenic genes. Moreover, DER and Htl are differentially involved in the specification of particular progenitors. We conclude that distinct cellular identity codes are generated by the combinatorial activities of Wg, Dpp, EGF, and FGF signals in the progressive determination of embryonic mesodermal cells.

Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides

The effects of pituitary-derived melanocortin peptides are primarily attributed to ACTH-mediated adrenocortical glucocorticoid production. Identification of a widely distributed receptor for ACTH/MSH peptides, the melanocortin-5 receptor (MC5-R), suggested non-steroidally mediated systemic effects of these peptides. Targeted disruption of the MC5-R produced mice with a severe defect in water repulsion and thermoregulation due to decreased production of sebaceous lipids. High levels of MC5-R was found in multiple exocrine tissues, including Harderian, preputial, lacrimal, and sebaceous glands, and was also shown to be required for production and stress-regulated synthesis of porphyrins by the Harderian gland and ACTH/MSH-regulated protein secretion by the lacrimal gland. These data show a requirement for the MC5-R in multiple exocrine glands for the production of numerous products, indicative of a coordinated system for regulation of exocrine gland function by melanocortin peptides.

Semaphorin-neuropilin interactions underlying sympathetic axon responses to class III semaphorins

Neuropilin-1 and neuropilin-2 show specificity in binding to different class III semaphorins, including Sema III, Sema E, and Sema IV, suggesting that the specificity of action of these semaphorins is dictated by the complement of neuropilins expressed by responsive neurons. In support of this, we show that sympathetic axons coexpress neuropilin-1 and -2, that their responses to Sema III, Sema E, and Sema IV are affected in predicted ways by antibodies to neuropilin-1, and that neuropilin-1 and -2 can form homo- and heterooligomers through an interaction involving at least partly the neuropilin MAM (meprin, A5, mu) domain. These results support the idea that in sympathetic axons, the Sema III signal is mediated predominantly by neuropilin-1 oligomers, the Sema IV signal by neuropilin-2 oligomers, and the Sema E signal by neuropilin-1 and -2, either as homo- or heterooligomers.

An essential role for retinoid receptors RARbeta and RXRgamma in long-term potentiation and depression

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) are the most widely studied forms of synaptic plasticity thought to underlie spatial learning and memory. We report here that RARbeta deficiency in mice virtually eliminates hippocampal CA1 LTP and LTD. It also results in substantial performance deficits in spatial learning and memory tasks. Surprisingly, RXRgamma null mice exhibit a distinct phenotype in which LTD is lost whereas LTP is normal. Thus, while retinoid receptors contribute to both LTP and LTD, they do so in different ways. These findings not only genetically uncouple LTP and LTD but also reveal a novel and unexpected role for vitamin A in higher cognitive functions.

Neurological illness in transgenic mice expressing a prion protein with an insertional mutation

Familial prion diseases are caused by mutations in the gene encoding the prion protein (PrP). We have produced transgenic mice that express the mouse homolog of a mutant human PrP containing a nine octapeptide insertion associated with prion dementia. These mice exhibit a slowly progressive neurological disorder characterized clinically by ataxia and neuropathologically by cerebellar atrophy and granule cell loss, gliosis, and PrP deposition that is most prominent in the cerebellum and hippocampus. Mutant PrP molecules expressed in the brains of these mice are resistant to digestion by low concentrations of proteinase K and display several other biochemical properties reminiscent of PrP(Sc), the pathogenic isoform of PrP. These results establish a new transgenic animal model of an inherited human prion disorder.

Formation and specification of ventral neuroblasts is controlled by vnd in Drosophila neurogenesis

During Drosophila neural development, neuroblasts delaminate from the neuroectoderm of each hemisegment in a stereotypic orthogonal array of five rows and three columns (ventral, intermediate, and dorsal). Prevailing evidence indicates that the individual neuroblast fate is determined by the domain-specific expression of genes along the dorsoventral and anteroposterior axis. Here, we analyze the role of Vnd, a NK-2 homeodomain protein, expressed initially in the ventral neuroectoderm adjacent to the ventral midline, in the dorsoventral patterning of the neuroectoderm and the neuroblasts. We show that in vnd null mutants most ventral neuroblasts do not form and the few that form do not develop ventral fates, but instead develop intermediate-like fates. Furthermore, we demonstrate that Vnd influences the gene expression patterns in the ventral proneural clusters and neuroectoderm, and that its action in neuroblast formation includes, but is not exclusive to the activation of proneural AS-C genes. Through the use of GAL4/UAS gene-expression system we show that ectopic Vnd expression can promote ventral-like fates in intermediate and dorsal neuroblasts and can suppress certain normal characteristics of the intermediate and dorsal neuroectoderm. Our results are discussed in the context of the current evidence in dorsoventral patterning in the Drosophila neuroectoderm.

A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal

Germ-line stem cells (GSCs) serve as the source for gametogenesis in diverse organisms. We cloned and characterized the Drosophila piwi gene and showed that it is required for the asymmetric division of GSCs to produce and maintain a daughter GSC but is not essential for the further differentiation of the committed daughter cell. Genetic mosaic and RNA in situ analyses suggest that piwi expression in adjacent somatic cells regulates GSC division. piwi encodes a highly basic novel protein, well conserved during evolution. We isolated piwi homologs in Caenorhabditis elegans and humans and also identified Arabidopsis piwi-like genes known to be required for meristem cell maintenance. Decreasing C. elegans piwi expression reduces the proliferation of GSC-equivalent cells. Thus, piwi represents a novel class of genes required for GSC division in diverse organisms.

Identification of the mouse neuromuscular degeneration gene and mapping of a second site suppressor allele

The nmd mouse mutation causes progressive degeneration of spinal motor neurons and muscle atrophy. We identified the mutated gene as the putative transcriptional activator and ATPase/DNA helicase previously described as Smbp2, Rip1, Gf1, or Catf1. Mutations were found in two alleles-a single amino acid deletion in nmdJ and a splice donor mutation in nmd2J. The selective vulnerability of motor neurons is striking in view of the widespread expression of this gene, although the pattern of degeneration may reflect a specific threshold since neither allele is null. In addition, the severity of the nmd phenotype is attenuated in a semidominant fashion by a major genetic locus on chromosome (Chr) 13. The identification of the nmd gene and mapping of a major suppressor provide new opportunities for understanding mechanisms of motor neuron degeneration.

Identification of a class of chromatin boundary elements

Boundary elements are thought to define the ends of functionally independent domains of genetic activity. An assay for boundary activity based on this concept measures the ability to insulate a bracketed, chromosomally integrated reporter gene from position effects. Despite their presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. The Drosophila protein BEAF binds the scs' boundary element of the 87A7 hsp70 locus and roughly half of polytene chromosome interband loci. To see if these sites represent a class of boundary elements that have BEAF in common, we have isolated and studied several genomic BEAF binding sites as candidate boundary elements (cBEs). BEAF binds with high affinity to clustered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to confer position-independent expression on a mini-white reporter gene in transgenic flies. Furthermore, point mutations in CGATA motifs that eliminate binding by BEAF also eliminate the ability to confer position-independent expression. Taken together, these findings suggest that clustered CGATA motifs are a hallmark of a BEAF-utilizing class of boundary elements found at many loci. This is the first example of a class of boundary elements that share a sequence motif and a binding protein.