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Presents a corrected version of the article 'Distinct cell killing properties of the Drosophila reaper, head involution defective, and grim genes,' published in a 1998 issue of 'Cell Death and Differentiation.'

Fine-needle aspiration (FNA) is the first-line and a cost-effective examination method of nonfunctional thyroid nodules. Acute transient thyroid swelling after an FNA is a rare complication, and to date, only 14 cases have been reported in the English literature. Herein, we report a case of a 26-year-old woman with acute transient thyroid swelling, which occurred after an ultrasound-guided FNA of a thyroid nodule. Although the patient had undergone an FNA without complication 2 years previously, the second FNA caused acute thyroid swelling. The present case emphasizes the potential risk of acute thyroid swelling associated with every FNA procedure., (© 2021 Wiley Periodicals LLC.)

Introduction: Afferent loop obstruction is an uncommon complication associated with Billroth II reconstruction or Roux-en-Y reconstruction after gastrectomy. Moreover, cases where the obstruction is caused by enterolith are rare. Here, we report a rare case of afferent loop obstruction caused by an enterolith after Roux-en-Y reconstruction of gastrectomy; subsequently, leading to ileus in the ileum., Presentation of Case: An 84-year-old man who received a Roux-en-Y distal gastrectomy for gastric cancer presented with symptoms of fever and jaundice 14 months later. Computed tomography (CT) scan revealed an enterolith in the duodenal afferent loop and a dilated intrahepatic bile duct. Although the obstructive jaundice and fever disappeared with conservative therapy, ileus occurred due to the movement of the enterolith into the ileum, which was refractory to conservative therapy. Therefore, enterotomy was performed to remove the enterolith, and the patient had an uneventful recovery. Histologically, the enterolith derived from food residue. No postsurgical sign of recurrence has been noted for 6 months., Conclusion: We report a rare case where an enterolith in a duodenal afferent loop after distal gastrectomy led to obstructive jaundice, and subsequently, caused ileus by its movement into the ileum., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Although appendicitis is a common disease, appendicitis concurrent with liver abscesses and sessile serrated adenoma/polyp (SSA/P) is rare., Presentation of Case: A 69-year-old man presented with symptoms of abdominal pain and fever. Computed tomography (CT) revealed multiple liver abscesses and an enlarged appendix with a pseudotumoral appearance, which suggested acute appendicitis. In the emergency operation, ileocecal resection was performed for the perforated appendicitis with an inflammatory mass in the ileocecum. On macroscopic examination, the torose lesion was localized at next to the appendiceal orifice. The tumor was diagnosed as SSA/P based on the microscopic finding. The postoperative course was uneventful, and the liver abscesses were cured by antibiotic therapy. The patient was discharged 17days after the surgery., Discussion: In this case, SSA/P localization at next to the appendiceal orifice was suggested as the cause of the perforated appendicitis with multiple liver abscesses. The patient was successfully treated with a combination of surgery and antibiotic therapy., Conclusion: This is the first reported case of a patient with SSA/P that led to acute appendicitis with multiple pyogenic liver abscesses., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: De Garengeot hernia is rare. Although previous reports have suggested various surgical options according to patient condition, comorbidities, surgeon preference, and clinical findings during surgery, a treatment strategy has not been established., Presentation of Case: An 81-year-old woman presented with an irreducible tender mass that was subsequently diagnosed as an incarcerated femoral hernia with a subcutaneous abscess in the right groin. Intraoperative findings revealed a necrotic and perforated appendix strangulated by the femoral ring for which an appendectomy and herniorrhaphy was performed concurrently through the hernia sac. The subcutaneous abscess cavity was washed thoroughly and a drainage tube was placed within it. The patient recovered uneventfully., Discussion: We suggest that the approach through the inguinal incision in both appendectomy and herniorrhaphy with drainage may be useful in avoiding intra-abdominal contamination in cases of de Garengeot hernia with subcutaneous abscess., Conclusion: Here, we described a case of de Garengeot hernia with a subcutaneous abscess in the groin. Clinicians should consider de Garengeot hernia in patients with a groin hernia, make an early diagnosis, and promptly provide surgical treatment to reduce the risk of complications., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Serum carbohydrate antigen 19-9 (CA 19-9), a marker of malignant tumors, is generally slightly elevated in benign conditions. We report a case of acute cholecystitis with a significantly elevated level of serum CA 19-9 based on positron emission tomography (PET)-computed tomography (CT) findings. A 65-year-old woman presented with abdominal pain and fever. A CT image revealed an enlarged gallbladder without tumor shadows. The C-reactive protein (CRP) level was elevated to 7.66 mg/dl. Moreover, the serum CA 19-9 level was significantly elevated to 19,392 U/ml. We started antibiotic treatment, because we suspected acute cholecystitis, but still, we could not ignore the possible presence of malignant tumors. After 11 days of antibiotic treatment, serum CRP and CA 19-9 levels decreased to 0.11 mg/dl and 1,049 U/ml, respectively. There was an accumulation of fluorine 18-labeled fluorodeoxyglucose (maximum standardized uptake value, 9.3) without tumor shadows in the liver, near the gallbladder, on the PET-CT examination. We considered the possibility that the inflammation had spread from the gallbladder to the liver, made a diagnosis of acute cholecystitis, and performed a cholecystectomy 33 days after treatment initiation. The serum CA 19-9 level decreased to 45 U/ml after the surgery. One year after the surgery, the patient was alive, and the serum CA 19-9 level was 34 U/ml. Acute cholecystitis with a significantly high elevation of the serum CA 19-9 level is rare. In such cases, it is important to confirm the change in the serum CA 19-9 level over time after antibiotic treatment and perform imaging studies to distinguish between inflammation and malignancy.

Anaplastic thyroid cancer is one of the most aggressive human malignancies and is resistant to multimodal treatments. The expression of h-prune, the human homologue of Drosophila prune, has been reported to be correlated with progression and aggressiveness in various cancers including breast, colorectal and pancreatic cancers. We examined the role of h-prune in anaplastic thyroid cancer cell migration, invasion and metastasis. Immunohistochemical analysis of h-prune was performed with 15 surgically resected specimens of anaplastic thyroid cancers. To investigate cell motility, Boyden chamber, wound healing and matrigel invasion assays were performed using cells from anaplastic thyroid cancer cell lines. A murine orthotopic thyroid cancer model was used to investigate metastatic ability. In the immunohistochemical analysis, only weak focal or no staining of h-prune was observed in non-tumor tissue. In contrast, diffuse staining of h-prune was observed in anaplastic thyroid cancer and lymph node metastasis samples. Both inhibition of h-prune phosphodiesterase activity with dipyridamole and small interfering RNA for h-prune suppressed 8505C and KTC-3 cell motility. In addition, treatment with dipyridamole and decreased expression of h-prune suppressed tumor invasion and pulmonary metastasis in a NOD/Shi-scid, IL-2Rγnull (NOG) mouse orthotopic thyroid cancer model. In conclusion, h-prune is frequently expressed in anaplastic thyroid cancer cells and lymph nodes metastasis, and promotes migration and invasion of anaplastic thyroid cancer cells and metastasis in an anaplastic thyroid cancer model. Thus, h-prune shows promise as a targeting candidate against anaplastic thyroid cancer.

We present the first case of central neurocytoma with a component suggesting ependymal-like differentiation as an unclassified glioneuronal tumor. The patient was a 26-year-old Japanese man with a brain tumor extending from the frontal wall of the bilateral lateral ventricles to the corpus callosum. Histologically, the tumor's neuronal component consisted of small bland looking cells with fine, delicate, neuropil-like processes forming a rosette structure; its glial component consisted of tumor cells with thick processes arranged around the thinly walled vessels, resulting in a perivascular pseudo-rosette formation and indicating ependymal-like differentiation. Immunohistochemically, the cytoplasm of the tumor cells with ependymal-like features was positive for glial fibrillary acidic protein and negative for synaptophysin, while the tumor component with neuronal features showed the opposite immunohistochemical staining pattern. Most of the tumor cells were positive for Olig2, but EMA, D2-40, CD99, p53, and mutant IDH-1 (R132H) were totally negative. Its Ki-67 labeling index was less than 1 %. Histologically, this tumor was diagnosed as a central neurocytoma with an ependymoma-like glial component, and its tumor grade was estimated at grade II. The tumor location, infiltrating the corpus callosum, and histology were distinctive and might represent a peculiar subtype of glioneuronal tumor.

Fatty liver (FL) is associated with development of hepatocellular carcinoma (HCC). However, whether FL itself promotes the progression of HCC is unclear. We recently found that hepatic stellate cells (HSCs) were prominently activated in the steatotic liver. Here, we investigated whether steatotic livers promote HCC progression and whether HSCs of steatotic liver are associated with HCC progression. We implanted rat HCC cells into diet-induced steatotic livers in rats via portal vein injection. Thereafter, HSCs and HCC cells were co-implanted subcutaneously into nude rats. Migration and proliferation of HCC cells were measured, and activation of ERK and Akt in these cells was determined by western blotting. Chemokines secreted from HSCs and HCC cells were also evaluated by ELISA. Steatotic livers significantly promoted HCC metastasis compared with non-steatotic livers. Additionally, co-implantation of HCC cells with HSCs from steatotic livers produced significantly larger tumors in recipient rats as compared to those induced by HCC cells co-implanted with HSCs from normal livers (NLs). HSCs isolated from steatotic livers, compared with HSCs isolated from NLs, secreted greater amounts of interleukin-1α, vascular endothelial growth factor, and transforming growth factor-β. These cytokines may enhance the proliferation and migration of HCC cells by increasing the phosphorylation of ERK and Akt in HCC cells. Moreover, we noted that the Rho-kinase inhibitor deactivated activated HSCs and attenuated HCC progression. In conclusion, the rat steatotic liver microenvironment favors HCC metastasis, and this effect appears to be promoted by activated HSCs in the steatotic liver., (© 2014 UICC.)

Two vanadium(V)-substituted tungsto-polyoxometalate isomers, 1- and 4-[S2VW17O62](5-), were prepared as their tetra-alkyl ammonium salts from a W(VI)-H2SO4-V(V) reaction mixture in aqueous CH3CN solution. X-ray crystallographic structural analysis revealed that both isomers have a Wells-Dawson-type structure with a higher occupancy of vanadium at polar sites and belt sites for 1- and 4-[S2VW17O62](5-), respectively. The isomers were also characterized by elemental analysis, infrared, Raman, UV-vis, and (51)V NMR spectroscopies as well as voltammetry, and the data obtained were compared with that derived from [S2W18O62](4-). Significantly, the reversible potentials for the vanadium(V/IV) couple for both 1- and 4-[S2VW17O62](5-) in CH3CN (0.1 M n-Bu4NPF6) are considerably more positive than the tungstate reduction process exhibited by the [S2W18O62](4-) framework, implying that the presence of vanadium should be useful in catalytic reactions. The one-electron-reduced [S2V(IV)W17O62](6-) forms of both isomers were prepared in solution by controlled potential bulk electrolysis and characterized by voltammetry and EPR spectroscopy.

Structures of the n-tetrabutylammonium salts of [SVM11O40](3-) (M = Mo, W) have been determined by X-ray crystallography and exhibit 3D networks with the V atom disordered over several sites. The cyclic voltammetric behavior of SVM11 in neutral and acidified acetonitrile solutions also has been investigated with respect to the V(V)/V(IV) couple. Results have been interpreted in conjunction with data provided by (51)V NMR spectroscopy on the oxidized V(V) form and by EPR spectroscopy on the reduced V(IV) form. Based on mechanistic details inferred from these studies, simulations of the cyclic voltammograms have been undertaken and results compared with experimental data in acidic media (two protonated forms) in order to provide estimates of equilibrium and kinetic parameters. For the V(V)/V(IV) couple in the series [XVM11O40](n-) (X = Si,Ge,P,As,S; M = Mo,W), the reversible potentials in neutral acetonitrile linearly depend on the total charge of the vanadium-substituted polyoxometalates, similar to the dependence previously reported for the non-substituted parent Keggin polyoxometalates [XM12O40](m-).

Purpose: To investigate the detectability of hepatocellular carcinoma (HCC) on Gd-EOB-enhanced MR images (Gd-EOB-MRI), we performed tumor-by-tumor analysis of pathologically confirmed tumors using explants from cirrhotic patients who had undergone liver transplantation., Materials and Methods: We studied 11 explanted livers and classified the tumor intensity during the arterial phase (AP) and the hepatobiliary phase (HBP) as low in HBP with early enhancement (EE) in AP (A), as high in HBP with EE in AP (B), as low in HBP without EE in AP (C), as high in HBP without EE in AP (D), and as iso-intense in HBP with EE in AP (E). The diagnostic criteria for HCC were (i) pattern A and C, (ii) pattern A and E, (iii) pattern C and E, and (iv) patterns A, C, and E., Results: Of the 71 HCCs, 22 were not detected at MRI; of these, 9 were moderately differentiated and 13 were well-differentiated HCCs. The sensitivity of Gd-EOB-MRI for diagnostic criteria 1, 2, 3, and 4 was 63.4%, 52.1%, 22.5%, and 69.0%., Conclusion: The maximum sensitivity of Gd-EOB-MRI for HCC was only 69.0% even when diagnostic criteria that included all previously reported HCC patterns were adopted., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Although several studies have shown that serum antithrombin III (ATIII) has anti-inflammatory effects, the prognostic value of ATIII in HCC is unknown. We investigated the influence of preoperative ATIII levels on the outcome of patients who underwent hepatectomy for hepatocellular carcinoma (HCC)., Methods: Data from 440 patients (314 patients with ATIII ≥ 70 % and 126 patients with ATIII <70 %) who underwent curative hepatectomy for HCC were retrospectively collected and analyzed. To overcome bias due to the different distribution of covariates for the 2 groups, propensity score matching was performed on the patients, and outcomes were compared., Results: The propensity score analysis revealed that 65 patients with ATIII of ≥ 70 % (group 1) and 65 patients with ATIII of <70 % (group 2) had the same preoperative and operative characteristics (excluding the ATIII level). The overall survival rate and the disease-free survival rate was significantly higher in group 1 than in group 2 (P = 0.005 and 0.011, respectively). Multivariate analysis showed that ATIII was a significant favorable factor for overall survival and disease-free survival of patients with HCC after curative hepatectomy., Conclusions: The prognosis of patients with HCC was found to be associated with preoperative antithrombin III levels. ATIII may be useful for predicting outcomes of patients with HCC after curative hepatectomy.

Background & Aims: Hepatic stellate cells are thought to play a role in modulating intrahepatic vascular resistance based on their capacity to contract via Rho signaling. We investigated the effect of a Rho-kinase inhibitor on ischemia-reperfusion injury in the steatotic liver., Methods: Steatotic livers, induced by a choline-deficient diet in rats, were subjected to ischemia-reperfusion injury. Hepatic stellate cells isolated from steatotic livers were analyzed for contractility and Rho signaling activity. The portal pressure of the perfused rat liver and the survival rate after ischemia-reperfusion were also investigated., Results: Hepatic stellate cells from steatotic livers showed increased contractility and upregulation of Rho-kinase 2 compared with those from normal livers. Furthermore, endothelin-1 significantly enhanced the contractility and phosphorylation level of myosin light chain and cofilin in hepatic stellate cells isolated from steatotic livers. A specific Rho-kinase inhibitor, fasudil, significantly suppressed the contractility and decreased the phosphorylation levels of myosin light chain and cofilin. Serum levels of endothelin-1 were markedly increased after IR in rats with steatotic livers, whereas fasudil significantly decreased endothelin-1 serum levels. Rats with steatotic livers showed a significant increase in portal perfusion pressure after ischemia-reperfusion and a significant decrease in survival rate; fasudil treatment significantly reduced these effects., Conclusions: Activation of Rho/Rho-kinase signaling in hepatic stellate cells isolated from steatotic livers is associated with an increased susceptibility to ischemia-reperfusion injury. A Rho-kinase inhibitor attenuated the activation of hepatic stellate cells isolated from steatotic livers and improved ischemia-reperfusion injury in steatotic rats., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The one electron electrochemical reduction of vanadium(V) substituted-Keggin type polyoxometalate anions [XV(V)M(11)O(40)](4-) (XV(V)M(11)) where X = P, As; M = Mo or W has been investigated in CH(3)CN as a function of acid concentration. EPR studies confirm that the product is the highly basic and hence readily protonated V(IV), (XV(IV)M(11)), species. In the absence of acid or in the presence of a large concentration excess, the V(V/IV) redox couple gives one well defined chemically and electrochemically reversible process under conditions of cyclic voltammetry. In contrast, three well resolved V(V/IV) processes are observed in the presence of a moderate concentration of acid in the case of XV(V)Mo(11) but only two with XV(V)W(11). NMR and EPR spectra have been obtained as a function of acid concentration when the polyoxometalate anions are in the V(V) and V(IV) oxidation levels respectively. All voltammetric and spectroscopic data indicate that protonation reactions are coupled to the V(V) and V(IV) redox chemistry but that the reduced V(IV) state is much more basic than the V(V) one and that (XV(IV)Mo(11)) is more basic than (XV(IV)W(11)). Digital simulations of voltammograms for reduction of XV(V)Mo(11) and XV(V)W(11) have been undertaken in CH(3)CN as a function of acid concentration in order to define the thermodynamics and kinetics associated with the V(V/IV) process and the equilibrium constants that accompany the coupled acid-base chemistry. EPR spectra allow an estimation of the relative concentrations of protonated species present in frozen glasses derived from one-electron bulk electrolysis and also allow inferences to be drawn on their nature. This study provides a far more detailed analysis of the coupling of proton and electron transfer than previously available.

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH(2)-terminal Reaper, Hid, Grim (RHG) motif. We have analyzed the importance of the RHG motifs in Reaper and Grim for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicated that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retained an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions were not efficiently blocked by the cell death inhibitors, Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, we identified a second region of sequence similarity in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities.

During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent on Single-minded (Sim), a basic-helix-loop-helix (bHLH)-PAS transcription factor. We show here that Fish-hook (Fish), a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression. single-minded, fish-hook, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays revealed genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Fish-hook directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Fish can form homodimers and also associates with other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes.

In Drosophila, the fish-hook (fish) gene encodes a Sox protein essential for embryonic segmentation and nervous system organization. In this study we examined potential functional roles of fish in postembryonic developmental processes, including those involved in adult appendage development. We show here that Fish protein is expressed in discrete patterns in the larval eye-antennal and leg imaginal discs, the central nervous system, the hindgut, and salivary glands. Genetic mosaic studies indicated that fish function is required for the growth or survival of imaginal cells, and the expression of engrailed and wingless. Ectopic expression of Fish protein resulted in severe disruption of adult structures; legs and antennae were truncated and eye formation was suppressed. These morphological defects were correlated with altered expression patterns of the wingless, decapentaplegic, and bric-a-brac genes. Finally, analysis of truncated versions of Fish protein indicated that the HMG domain was sufficient for Fish nuclear localization and that removal of the transcriptional activation domain did not eliminate Fish function. While Sox proteins have been shown to be important for eye and limb formation in vertebrates, these data provide the first evidence for Sox protein functions in appendage development in invertebrates., (Copyright 2000 Academic Press.)

We used the abdominal intersegmental muscles (ISMs) of the moth Manduca sexta as a source of transcripts that are dramatically up-regulated during programmed cell death. One of these transcripts, Small Cytoplasmic Leucine-Rich Repeat Protein (SCLP), encodes a protein of approximately 24 kD that contains four perfect and two imperfect leucine-rich repeat (LRR) motifs. DNA sequence database analysis suggests that SCLP is a phylogenetically-conserved gene of unknown function. Both Northern and Western blots demonstrated that SCLP is expressed in the ISMs at all stages examined, but increases greater than 10-fold when the cells become committed to die. This increase in expression is regulated by the same change in the circulating ecdysteroid titer that controls death. Low levels of SCLP expression are also seen in flight muscle and fat body, but not in ovary, male sexual accessory gland, or Malpighian tubules. Immunohistochemical analysis demonstrates that SCLP is a cytoplasmic protein. Western blot analysis of proteins from the fly Drosophila suggests that an SCLP-related protein is expressed at the larval and pupal stages, but not in embryos or adults. Targeted expression of moth SCLP to a variety of different tissues in Drosophila using the Gal4/UAS P element system failed to generate an overt phenotype. These data are interpreted as suggesting that whereas SCLP presumably plays an important role in programmed cell death of muscle, perhaps by acting as an adaptor protein, its expression is insufficient to initiate death by itself., (Copyright 1999 John Wiley & Sons, Inc.)

During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of DALP, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth Manduca sexta become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C(2)C(12) myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved "switches" to block muscle differentiation and induce death.

Human neurodegenerative disorders are typified by late onset cell loss in specific brain regions and stereotypic neuroanatomical and behavioral aberrations. Recent studies suggest that molecular genetic approaches in Drosophila may shed important new light on conserved mechanisms underlying such disorders.

The Drosophila reaper, head involution defective (hid), and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. In this study we use the Gal4/UAS targeted gene expression system to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. We also show that in both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with reaper or hid to induce higher levels of midline cell death than observed for any of the genes individually. Finally we analyzed the function of a truncated Reaper-C protein which lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C revealed cell killing activities distinct from full length Reaper, and indicated that the conserved NH2-terminal domain acts in part to modulate Reaper activity.

In this study we investigate the gene regulatory functions of Drosophila Fish-hook (Fish), a high mobility group (HMG) Sox protein that is essential for embryonic segmentation. We show that the Fish HMG domain binds to the vertebrate Sox protein consensus DNA binding sites, AACAAT and AACAAAG, and that this binding induces an 85 degrees DNA bend. In addition, we use a heterologous yeast system to show that the NH2-terminal portion of Fish protein can function as a transcriptional activator. Fish directly regulates the expression of the pair rule gene, even-skipped (eve), by binding to multiple sites located in downstream regulatory regions that direct formation of eve stripes 1, 4, 5, and 6. Fish may function along with the Drosophila POU domain proteins Pdm-1 and Pdm-2 to regulate eve transcription, as genetic interactions were detected between fish and pdm mutants. Finally, we determined that Fish protein is expressed in a dynamic pattern throughout embryogenesis, and is present in nuclear and cytoplasmic compartments., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved.)

We have investigated the roles of the single-minded (sim) and rhomboid (rho) genes in generating distinct cell fates in the Drosophila embryonic neuroectoderm. We show that sim functions to repress ventral ectodermal cell fates, as in sim mutants mesectodermal cells adopt the fates of neighboring ventral ectodermal cells and targeted sim expression in P[paired.Gal4]/P[UAS-sim] embryos results in loss of epidermal cells. We also find that rho is not required for early expression of sim or ventral nervous system defective in mesectodermal or ventral ectodermal cells; targeted rho expression in P[paired-Gal4]/P[UAS-rho] embryos results in lateral-to-ventral cell fate shifts in the developing neuroectoderm; and midline-targeted rho expression can rescue the medial denticle fusions in rho mutant cuticles.

The Drosophila melanogaster (Dm) similar (sima) gene was isolated using a low-stringency hybridization screen employing a Dm single-minded gene basic helix-loop-helix (bHLH) DNA probe. sima is a member of the bHLH-PAS gene family and the conceptual protein shares a number of structural features, including a bHLH domain, PAS domain, and homopolymeric amino acid stretches. Sima is most closely related to the human hypoxia-inducible factor 1 alpha bHLH-PAS protein. In situ hybridization experiments reveal that sima is transcribed in most or all cells throughout embryogenesis. It has been cytologically mapped to position 99D on the third chromosome, and is not closely linked to other known bHLH-PAS genes.

Background: During the development of the central nervous system, large numbers of cells die by programmed cell death. This process requires the activity of specific gene products and subserves functions that include regulating the sizes of interacting cell populations and removing cells that provide transient functions. Resolution of programmed cell death often involves the elimination of dying cell corpses by phagocytic macrophages. In Drosophila, the reaper gene plays a crucial role in mediating programmed cell death; chromosomal deficiencies which remove reaper result in an absence of programmed cell death. We have used a reaper-deficiency mutant strain Df(3R)H99 (or H99), in conjunction with strains containing cell-type-specific markers, to examine the role of programmed cell death in differentiation of the embryonic central nervous system midline., Results: Midline cell death was identified both by the presence of excess midline cells in H99 mutants and by the engulfment of dying midline cells by macrophages in wild-type embryos. These developmental deaths are lineage-specific: prominent midline glial death was observed, while little if any death was detected among the ventral unpaired median neurons. Examination of H99 mutants indicates that cell death is not required for the formation of macrophage precursors, or for their subsequent migration throughout the embryo; however, in the absence of dying cells, macrophage precursors do not exhibit morphological differentiation or phagocytosis. In both wild-type and H99 mutant embryos, a subset of macrophages migrate along the ventral midline. This midline migration is not observed in single-minded mutants, in which ventral midline cells fail to develop., Conclusions: Programmed cell death plays a crucial role in the development of the central nervous system midline, and dying midline cells are rapidly eliminated by phagocytic macrophages. It seems that the generation of engulfment signals in cells undergoing programmed cell death is downstream of reaper gene function, and that central nervous system midline and/or ventral epidermal cells provide directional cues for migrating macrophages.

1. The midline cells of the Drosophila embryonic CNS comprise a discrete neuroanatomical structure consisting of a small subset of neurons and glia. 2. Developmental commitment of the CNS midline cells requires the action of dorsal/ventral patterning genes. 3. The single-minded gene encodes a basic-helix-loop-helix transcription factor and acts as a master regulator for the CNS midline lineage. 4. A number of different transcription factors and proteins involved in cell-cell interactions are necessary for the differentiation of midline neurons and glia. 5. CNS midline cells have important functions in the formation of the ventral epidermis and axon commissures.

Our goal is to understand the molecular mechanisms that govern the formation of the central nervous system. In particular, we have focused on the development of a small group of neurons and glia that lie along the midline of the Drosophila CNS. These midline cells possess a number of unique attributes which make them particularly amenable to molecular, cellular, and genetic examinations of nervous system formation and function. In addition, the midline cells exhibit distinctive ontogeny, morphology, anatomical position, and patterns of gene expression which suggest that they may provide unique functions to the developing CNS. The single-minded gene encodes a nuclear protein which is specifically expressed in the midline cells and has been shown to play a crucial role in midline cell development and CNS formation. Genetic experiments reveal that sim is required for the expression of many CNS midline genes which are thought to be involved in the proper differentiation of these cells. In order to identify additional genes which are expressed in some or all of the midline cells at different developmental stages, a technique known as enhancer trap screening was employed. This screen led to the identification of a large number of potential genes which exhibit various midline expression patterns and may be involved in discrete aspects of midline cell development. Further molecular, genetic, and biochemical analyses of sim and several of the enhancer trap lines are being pursued. This should permit elucidation of the genetic hierarchy which acts in the specification, differentiation, and function of these CNS midline cells.

Transcription of the egg-laying hormone (ELH) gene family was examined by characterizing homologous cDNA clones from abdominal ganglion and atrial gland cDNA libraries. All cDNAs contain an exon that spans the coding region (exon III) and one or two additional exons. The tissue-specific expression of the ELH gene family was confirmed by the observation that exon III encodes the ELH precursor protein in the bag cell transcripts and either the A or B precursor proteins in the atrial gland transcripts. The cDNA clones also contain 5' untranslated exons not present in the previously isolated genomic clones. One type of transcript has a 40-base pair segment, designated exon I, contiguous with exon III. A second type of transcript has an additional 149 base pairs of DNA, designated exon II, located between exons I and III. Several genomic clones containing exons I and II were isolated. DNA sequence analysis reveals that exons I and II are directly linked and that they are separated from exon III by an intervening sequence of at least 5 kilobases (kb). Consensus sequences for a putative promotor region and also for RNA splicing and polyadenylation were identified. From this work we can describe a prototype ELH gene complete with identified sequences necessary for the proper initiation of transcription and the subsequent processing of the transcript.

Evolution of the egg-laying hormone (ELH) gene family was examined in the genus Aplysia using genomic Southern blotting, gene cloning, and immunocytochemical techniques to identify and characterize homologous sequences. Most of the species examined have fewer than the 4-5 ELH-related genes present in the A. californica genome (Mahon et al., 1985; Scheller et al., 1983). In A. parvula there are 2 ELH genes, and unlike A. californica, no sequences were found to encode the A or B peptides. The 2 A. parvula ELH genes share at least 90% DNA sequence homology, while the homology between the A. parvula and A. californica ELH genes is 71%. The structural organization of the A. parvula ELH precursor is quite similar to the ELH precursor of A. californica, with all but one of the potential proteolytic cleavage sites conserved. The overall amino acid homology between the A. parvula and A. californica ELH precursors is 66%; however, the alpha and beta bag cell peptides, as well as ELH, are more highly conserved, suggesting that these peptides have important physiological and behavioral roles within both Aplysia species. Immunocytochemical studies indicate that the A. parvula ELH genes are expressed in 2 bag-cell-like clusters of about 40 neurons each in the abdominal ganglion. There does not appear to be an atrial gland in A. parvula; however, ELH-immunoreactive peripheral neurons and their processes are observed along the perimeter of the large hermaphroditic duct.

The Aplysia neuroendocrine system is a particularly advantageous model for cellular and molecular studies because of the relatively small number and large size of its component neurons. Recombinant DNA techniques have been used to isolate the genes that encode the precursors of peptides expressed in identified neurons of known function. The organization and developmental expression of these genes have been examined in detail. Several of the genes encode precursors of multiple biologically active peptides that are expressed in cells which also contain classical transmitters. These studies, as well as immunohistochemical studies and the use of intracellular recording and voltage clamp techniques are the first steps toward revealing the mechanisms by which neuropeptides govern simple behaviors.

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.

RNA blotting and cDNA cloning techniques were used to study expression of the egg-laying hormone (ELH) gene family in the head ganglia of Aplysia californica. All head ganglia were found to express a 1800 nucleotide (nt) mRNA homologous to the ELH gene family. The nucleotide sequence of a clone isolated from a ring ganglia cDNA library demonstrates that this message encodes the ELH precursor. Further studies demonstrate the presence of a smaller, 1500 nt, transcript which encodes the peptide A precursor. However, the level of this mRNA is at least 10-fold lower than the ELH encoding message.