Evidence that preaxial polydactyly in the Doublefoot mutant is due to ectopic Indian Hedgehog signaling

Patterning of the vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) located at the posterior limb margin. One of the vertebrate Hh family members, Shh, has been shown to be able to mediate the function of the ZPA. Several naturally occurring mouse mutations with the phenotype of preaxial polydactyly exhibit ectopic Shh expression at the anterior limb margin. In this study, we report the molecular characterization of a spontaneous mouse mutation, Doublefoot (Dbf). Dbf is a dominant mutation which maps to chromosome 1. Heterozygous and homozygous embryos display a severe polydactyly with 6 to 8 digits on each limb. We show here that Shh is expressed normally in Dbf mutants. In contrast, a second Hh family member, Indian hedgehog (Ihh) which maps close to Dbf, is ectopically expressed in the distal limb bud. Ectopic Ihh expression in the distal and anterior limb bud results in the ectopic activation of several genes associated with anterior-posterior and proximal-distal patterning (Fgf4, Hoxd13, Bmp2). In addition, specific components in the Hedgehog pathway are either ectopically activated (Ptc, Ptc-2, Gli1) or repressed (Gli2). We propose that misexpression of Ihh, and not a novel Smoothened ligand as recently suggested (Hayes et al., 1998), is responsible for the Dbf phenotype. We consider that Ihh has a similar activity to Shh when expressed in the early Shh-responsive limb bud. To determine whether Dbf maps to the Ihh locus, which is also on chromosome 1, we performed an interspecific backcross. These results demonstrate that Dbf and Ihh are genetically separated by approximately 1.3 centimorgans, suggesting that Dbf mutation may cause an exceptionally long-range disruption of Ihh regulation. Although this leads to ectopic activation of Ihh, normal expression of Ihh in the cartilaginous elements is retained.     

Mutations of the RET-GDNF signaling pathway in Ondine''s curse

Sarcoidosis is a multi-systemic granulomatous disease of unknown cause. It commonly involves lymph nodes, lungs, eyes, and skin. Cardiac sarcoid may be isolated, or associated with systemic involvement. Cardiac involvement is found in 20-50% of autopsied patients with sarcoidosis. However, it only gives rise to clinical manifestations in about 5% of patients. Cardiac involvement by sarcoid has been reported to manifest as complete heart block, papillary muscle dysfunction, congestive heart failure, pericarditis and/or effusion, conduction abnormality or arrhythmia, chest pain, and sudden death. The most common site of involvement is the interventricular septum base, which when involved may lead to heart block or arrhythmia. We report a case of sudden death in a 33-year-old male with a history of surgically repaired congenital heart disease. Although his congenital heart disease was originally postulated to be important in his death, autopsy examination revealed cardiac sarcoid with prominent involvement of the conduction system.     

Hedgehog and beyond

The authors describe a case of an epidermoidal cyst of the spleen, which they were able to observe in an emergency case, due to suppuration of the cyst. Primary cysts of the spleen represent a rare dysontegenetic pathological condition which is, for this reason, often underestimated. The general clinical summary is related here, with particular reference to supporative complications, which cause problema of differential diagnosis with patients suffering from abscess of the spleen. The diagnosis can be made as a result of standard X-ray procedures and computed tomography and above all, following a simple scan. The treatment recommended in this case is an open splenectomy, in order to check the allarming clinical symptoms of the patient, which can deteriorate. The use of laparoscopical techniques or trans-cutaneous draining is reserved for salected cases.     

Cellular interactions in the thymus regulate the protein kinase C signaling pathway

Lung cancers occur more commonly in the upper lobes than in the lower lobes, but its pathophysiologic basis is not well understood. Because numerous studies have reported a consistent inverse relationship between lung cancer risk and intake of certain vegetables and fruits, we hypothesized that the balance between diet-derived protective substances delivered via the circulation and cigarette-derived carcinogenic substances delivered via the airways would be less favorable in the upper lobes compared with the lower lobes, hence accounting for the upper lobe predominance of tumors among smokers. Thus, we examined the association between diet and tumor location in 328 patients with lung cancer. The ratio of upper to lower lobe tumors was 2.5:1.0. In univariate analysis, age, height, weight, sex, race, family history of cancer, education level, tumor histology, calories consumed per day, and intake of animal fat did not differ significantly between patients with upper versus lower lobe tumors. Predictors of tumor location in univariate analysis were family history of lung cancer; smoking history; history of asbestos exposure; and intakes of yellow-orange vegetables, alpha-carotene, beta-carotene, and vitamins A, C, and E. In multivariable logistic regression analysis, the independent predictors of upper lobe tumor location were family history of lung cancer (p = 0.03), history of asbestos exposure (p = 0.02), less intake of yellow-orange vegetables (p < 0.04), and less intake of vitamin E (p = 0.05). Our results show a strong inverse association between upper lobe location of lung cancer and intake of yellow-orange vegetables and vitamin E.     

IL-16 activates the SAPK signaling pathway in CD4+ macrophages

IL-16 has been reported as a modulator of T cell activation and was shown to function as chemoattractant factor. The chemotactic activity of IL-16 depends on the expression of CD4 on the surface of target cells, but the intracellular signaling pathways are only now being deciphered. This report describes IL-16 as an additional activator of the stress-activated protein kinase (SAPK) pathway in CD4+ macrophages. Treatment of these cells with recombinant expressed IL-16 leads to the phosphorylation of SEK-1, resulting in activation of the SAPKs p46 and p54. IL-16 stimulation also leads to the phosphorylation of c-Jun and p38 MAPK (mitogen-activated protein kinase), without inducing MAPK-family members ERK-1 and ERK-2. Interestingly, the IL-16-mediated activation of SAPKs and p38 MAPK in macrophages alone induces no detectable apoptotic cell death. These observations suggest specific regulatory functions of IL-16 distinct from the proinflammatory cytokines TNF-alpha and IL-1beta.     

Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway

IL-1 plays an important role in the pathophysiologic responses to infection and inflammation, in part by mediating its own production and that of other proinflammatory cytokines. However, the relative contribution of IL-1 alpha and IL-1 beta to the inflammatory response has not been well clarified. Using IL-1 beta-deficient (IL-1 beta -/-) mice, we investigated the specific role of IL-1 beta in the in vivo and in vitro response to LPS. No differences between IL-1 beta +/+ and IL-1 beta -/- mice were observed in circulating levels for IL-1 alpha, IL-6, or TNF-alpha after the systemic administration of either a low (5 micrograms/kg) or high (5 mg/kg) dose of LPS. IL-1 beta -/- mice also had a normal response to LPS in terms of activation of the hypothalamus-pituitary-adrenal axis, hypoglycemia, serum amyloid A production, and anorexia. IL-1 beta -/- mice were normally sensitive to the lethal effect of LPS and were protected against LPS toxicity when pretreated with low-dose LPS. However, in vitro, peritoneal macrophages from IL-1 beta -/- mice stimulated with LPS produced significantly less IL-1 alpha than macrophages from IL-1 beta +/+ mice (p < 0.05). No differences in IL-6 or TNF-alpha synthesis were observed between macrophages from IL-1 beta +/+ and IL-1 beta -/- mice. In summary, our results suggest that either IL-1 beta is not essential for the in vivo systemic response to LPS or that its role can be fulfilled by other cytokines with overlapping activities.     

Low salt intake down-regulates the guanylin signaling pathway in rat distal colon

BACKGROUND & AIMS: Guanylin, an endogenous gastrointestinal peptide, causes the translocation of NaCl from interstitial fluid to the intestinal lumen. The aim of this study was to examine whether changes in dietary salt intake lead to compensatory changes in expression of the guanylin signaling pathway. METHODS: Rats received low-, normal-, or high-sodium diets for 1 week. Colonic guanylin expression was evaluated by Western and Northern blotting, rates of guanylin secretion by measuring biologically active guanylin released into the medium from colon explants, and expression of the guanylin receptor (C-type guanylate cyclase) by Northern blotting and bioassay. RESULTS: By every criterion, the low-salt diet reduced expression of guanylin to 30%-40% of the level found in control animals. Guanylin receptor expression was also decreased, although less dramatically and with a lower statistical significance. For both guanylin and guanylin receptor, the high-salt diet had no significant effect on expression. CONCLUSIONS: The data support the hypothesis that the guanylin pathway is down-regulated as an adaptive response to salt restriction.     

Hedgehog rehabilitation in perspective

Thousands of hedgehogs are taken into care each year, mostly injured animals or late-born young unlikely to survive hibernation. Many are returned to the wild, but until recently there was little information about their welfare, behaviour or survival after release. A review of three studies undertaken in different regions of Britain, showed that the majority of 33 released adults and juveniles adapted quickly to life in the wild despite the juveniles having been raised in captivity. They found food readily, made nests and rapidly learned their way about. They all lost weight initially but, after two three weeks, stabilised at a similar weight to wild animals. The hedgehogs which were heaviest at release lost the greatest percentage of their bodyweight, suggesting they had become overweight in captivity. The hedgehogs which were known not to have survived at least six weeks after release included three road casualties and three eaten by badgers. Although wild hedgehogs may die in the same ways, it is possible that the released hedgehogs' susceptibility to such dangers may be increased by their becoming tame during captivity. However, the high survival rate suggests that the release of rehabilitated hedgehogs back into the wild is worthwhile.     

A novel signaling pathway controlling induced systemic resistance in Arabidopsis

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P. fluorescens WCS417r-mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid-nonaccumulating NahG plants. Moreover, methyl jasmonate-induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate-induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.     

A frizzled homolog functions in a vertebrate Wnt signaling pathway

BACKGROUND: Wnts are secreted proteins implicated in cell-cell interactions during embryogenesis and tumorigenesis, but receptors involved in transducing Wnt signals have not yet been definitively identified. Members of a large family of putative transmembrane receptors homologous to the frizzled protein in Drosophila have been identified recently in both vertebrates and invertebrates, raising the question of whether they are involved in transducing signals for any known signaling factors. RESULTS: To test the potential involvement of frizzled homologs in Wnt signaling, we examined the effects of overexpressing rat frizzled-1 (Rfz-1) on the subcellular distribution of Wnts and of dishevelled, a cytoplasmic component of the Wnt signalling pathway. We demonstrate that ectopic expression of Rfz-1 recruits the dishevelled proten-as well as Xenopus Wnt-8 (Xwnt-8), but not the functionally distinct Xwnt-5A-to the plasma membrane. Moreover, Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes, siamois and Xnr-3, in Xenopus explants in a manner which is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together in this assay, we observe greater induction of these genes, indicating that Rfz-1 can synergize with a Wnt. CONCLUSIONS: The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner which discriminates between functionally distinct Wnts, which involves translocation of the dishevelled protein to the plasma membrane, and which works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex.     

The c-kit receptor and its possible signaling transduction pathway in mouse spermatozoa

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.     

Cortactin-Src kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway.     

Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway

Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.     

A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway

Continuous growth and development in plants are accomplished by meristems, groups of undifferentiated cells that persist as stem cells and initiate organs. While the structures of the apical and floral meristems in dicotyledonous plants have been well described, little is known about the underlying molecular mechanisms controlling cell proliferation and differentiation in these structures. We have shown previously that the CLAVATA1 (CLV1) gene in Arabidopsis encodes a receptor kinase-like protein that controls the size of the apical and floral meristems. Here, we show that KAPP, a gene encoding a kinase-associated protein phosphatase, is expressed in apical and young floral meristems, along with CLV1. Overexpression of KAPP mimics the clv1 mutant phenotype. Furthermore, CLV1 has kinase activity: it phosphorylates both itself and KAPP. Finally, KAPP binds and dephosphorylates CLV1. We present a model where KAPP functions as a negative regulator of the CLAVATA1 signal transduction pathway.     

Opposing actions of CSW and RasGAP modulate the strength of Torso RTK signaling in the Drosophila terminal pathway

The NMDA receptor antagonistic effects of budipine were assessed using concentration- and patch-clamp techniques on cultured striatal, hippocampal, cortical and superior colliculus neurones. Inward current responses of striatal neurones to NMDA (200 microM) at -70 mV were antagonized by budipine in a concentration-dependent manner (50% inhibitory concentration (IC50) 59.4 +/- 10.7 microM, n = 17) with 24 times lower potency than memantine but similar potency to amantadine. In striatal neurones, budipine blocked outward currents at +70 mV with an IC50 of 827 microM, suggesting that the binding site is less deep in the channel (delta = 0.45) than for memantine. However, more detailed analysis of the fractional block by budipine 300 microM in hippocampal neurones gave a delta-value of 0.90, but revealed that 28% block is mediated at a voltage-independent site. This voltage-insensitive site was accessible in the absence of agonist. Budipine exhibited concentration-dependent open channel blocking kinetics (kappa(on) = 0.71 x 10(4) M(-1) s(-1)) whereas the fast offset rate was concentration-independent (kappa(off) = 0.63 s(-1)). Calculation of the ratio kappa(off)/kappa(on) revealed an apparent Kd value of 88.7 microM. Budipine, memantine and amantadine had similar effects against NMDA-induced currents in cultured hippocampal, cortical and superior colliculus neurones, although amantadine was somewhat more potent in cultured striatal neurones. The relevance of NMDA receptor antagonism to the anti-Parkinsonian effects of budipine remains to be established.