Acute appendicitis presenting as a perinephric abscess.
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Abscess↗
Biomedical subjects
J A Porter
Publications and source records attributed to J A Porter.
A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog.
Within the past few years, members of the hedgehog (hh) family of secreted signalling proteins have emerged as the primary signals generated by certain embryonic patterning centres. In vertebrate embryos, for example, sonic hedgehog expression in the notochord appears to be responsible for the local and long-range induction of ventral cell types within the neural tube and somites (reviewed in refs 1, 2). Protein products encoded by hh family members are synthesized as precursors that undergo autoprocessing to generate an amino-terminal domain that appears to be responsible for both local and long-range signalling activities, and a carboxy-terminal domain that contains the autoprocessing activity. As part of an effort to understand how hh family members participate in cell-to-cell signalling, we have determined and report here the crystal structure at 1.7 A of the amino-terminal domain of murine Sonic hedgehog (Shh-N). The structure revealed a tetrahedrally coordinated zinc ion that appears to be structurally analogous to the zinc coordination sites of zinc hydrolases, such as thermolysin and carboxypeptidase A. This previously unsuspected catalytic site represents a distinct activity from the autoprocessing activity that resides in the carboxy-terminal domain.
Amino Acid Sequence↗
Calmodulin binding to Drosophila NinaC required for termination of phototransduction.
The ninaC locus encodes two unconventional myosins, p132 and p174, consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC are the major calmodulin-binding proteins in the retina and the NinaC-calmodulin interaction is required for the normal subcellular localization of calmodulin as well as for normal photo-transduction. In the current report, we present evidence for two calmodulin-binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 was found to be common to both p132 and p174 while C2 was unique to p174. To address the requirements for calmodulin binding at each site in vivo, we generated transgenic flies expressing ninaC genes deleted for either C1 or C2. We found that the spatial localization of calmodulin depended on binding to both C1 and C2. Furthermore, mutation of either site resulted in a defective photoresponse. A prolonged depolarization afterpotential (PDA) was elicited at lower light intensities than necessary to produce a PDA in wild-type flies. These results suggest that calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction.
Amino Acid Sequence↗
Patterning activities of vertebrate hedgehog proteins in the developing eye and brain.
BACKGROUND: The hedgehog (hh) family of secreted signaling proteins is responsible for developmental patterning in a variety of systems, including the neural tube, limbs and somites. Within the neural tube, at the level of the spinal cord, products of the vertebrate gene sonic hedgehog (shh) are proposed to function as a ventral patterning influence, with the capability of inducing floor plate and motor neurons. RESULTS: We report the isolation of tiggy-winkle hedgehog (twhh), a novel member of the zebrafish hh gene family. Both twhh and shh are expressed in the ventral midline of the embryonic zebrafish neural tube and brain, but twhh expression becomes limited to the neural tube, whereas shh is also expressed in the notochord. Both genes are expressed in the developing brain, in domains that include a discrete region in the floor of the diencephalon, located between the sites of the future optic stalks. Using pax-2 and pax-6 as markers of proximo-distal fate within the developing eye, we found that ectopic expression of either hh gene promoted proximal fates and suppressed distal fates. In contrast, proximal fates were lost in cyclops mutant embryos, which lack twhh- and shh-expressing forebrain cells. Both twhh and shh proteins undergo autoproteolytic processing in vivo; a fragment corresponding to the amino-terminal cleavage product was sufficient to carry out all signaling activities associated with twhh in eye and brain development. CONCLUSIONS: These findings suggest that secreted signals encoded by members of the hedgehog gene family, emanating from the ventral midline of the neural tube, not only play important roles in dorso-ventral patterning of the brain but also appear to constitute an early patterning activity along the proximo-distal axis of the developing eyes.
Amino Acid Sequence↗
Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage.
BACKGROUND: Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS: We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS: Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.
Animals↗
Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis.
The differentiation of floor plate cells and motor neurons can be induced by Sonic hedgehog (SHH), a secreted signaling protein that undergoes autoproteolytic cleavage to generate amino- and carboxy-terminal products. We have found that both floor plate cells and motor neurons are induced by the amino-terminal cleavage product of SHH (SHH-N). The threshold concentration of SHH-N required for motor neuron induction is about 5-fold lower than that required for floor plate induction. Higher concentrations of SHH-N can induce floor plate cells at the expense of motor neuron differentiation. Our results suggest that the induction of floor plate cells and motor neurons by the notochord in vivo is mediated by exposure of neural plate cells to different concentrations of the amino-terminal product of SHH autoproteolytic cleavage.
Animals↗
Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway.
A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. Long-range effects of hedgehog (hh) signaling have been suggested to result either from local induction of a secondary diffusible signal or from the direct action of the highly diffusible carboxy-terminal product of HH autoproteolytic cleavage. Here we provide evidence that the long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product. We also show that pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. Our results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members.
Animals↗
The product of hedgehog autoproteolytic cleavage active in local and long-range signalling.
The secreted protein products of the hedgehog (hh) gene family are associated with local and long-range signalling activities that are responsible for developmental patterning in multiple systems, including Drosophila embryonic and larval tissues and vertebrate neural tube, limbs and somites. In a process that is critical for full biological activity, the hedgehog protein (Hh) undergoes autoproteolysis to generate two biochemically distinct products, an 18K amino-terminal fragment, N, and a 25K carboxy-terminal fragment, C (ref. 16); mutations that block autoproteolysis impair Hh function. We have identified the site of autoproteolytic cleavage and find that it is broadly conserved throughout the hedgehog family. Knowing the site of cleavage, we were able to test the function of the N and C cleavage products in Drosophila assays. We show here that the N product is the active species in both local and long-range signalling. Consistent with this, all twelve mapped hedgehog mutations either affected the structure of the N product directly or otherwise blocked the release of N from the Hh precursor as a result of deletion or alteration of sequences in the C domain.
Amino Acid Sequence↗
Induction of midbrain dopaminergic neurons by Sonic hedgehog.
Midbrain dopaminergic neurons, whose loss in adults results in Parkinson's disease, can be specified during embryonic development by a contact-dependent signal from floor plate cells. Here we show that the amino-terminal product of Sonic hedgehog autoproteolysis (SHH-N), an inductive signal expressed by floor plate cells, can induce dopaminergic neurons in vitro. We show further that manipulations to increase the activity of cyclic AMP-dependent protein kinase A, which is known to antagonize hedgehog signaling, can block dopaminergic neuron induction by floor plate cells. Our results and those of other studies indicate that SHH-N can function in a dose-dependent manner to induce different cell types within the neural tube. Our results also provide the basis for a potential cell transplantation therapy for Parkinson's disease.
Animals↗
Predictive factors for early mortality after percutaneous endoscopic gastrostomy.
BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) is a safe access procedure for enteral nutrition. The purpose of this investigation is to identify predictive factors for early mortality after PEG. METHODS: A retrospective review of the hospital records of 416 patients undergoing PEG from June 1, 1989, through December 31, 1991, was conducted. Patient demographics, admitting diagnosis, indication for PEG, risk factors for early mortality, and cause and date of death were reviewed. Logistic regression analysis was used to develop a model to predict early mortality after PEG. The follow-up period ranged from 1 to 30 months. RESULTS: The overall mortality rate in this review was 227 of 416 patients (54.6%). The 7- and 30-day case fatality rates were 39 of 416 (9.4%) and 97 of 416 (23.3%), respectively. Logistic regression analysis showed that urinary tract infection (odds ratio (OR) = 3.05; 95% confidence interval (CI) = 1.45-6.43) and previous aspiration (OR = 6.86; 95% CI = 3.27-14.4) were predictive factors for death at 1 week after PEG. Patients who had both risk factors had a 48.4% probability of dying within 7 days after PEG insertion, whereas those who had no risk factors had a 4.3% probability of death. Urinary tract infection (OR = 2.00; 95% CI = 1.17-3.41), previous aspiration (OR = 3.62; 95% CI = 2.00-6.55), and age greater than 75 years (OR = 2.49; 95% CI = 1.47-4.21) were predictive factors for death at 1 month after PEG. Patients who had all three risk factors had a 67.1% probability of death at 1 month while those who had no risk factors had a 10% probability of death. CONCLUSIONS: A subgroup of patients exists that has a very high mortality rate after PEG. Less invasive ways of nutritionally supporting these high-risk patients should be evaluated.
Adolescent↗
Neuroreality. I. Dedicated demolition of the decade of the brain:the genuine threat to neurologic research from the animal radical right American Academy of Neurology Animal Studies Subcommitte.
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Animal Rights↗
Neuroreality I. Dedicated demolition of the Decade of the Brain: the genuine threat to neurologic research from the animal radical right. The American Academy of Neurology Animal Studies Subcommittee.
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Animal Rights↗
Autoproteolysis in hedgehog protein biogenesis.
Extracellular signaling proteins encoded by the hedgehog (hh) multigene family are responsible for the patterning of a variety of embryonic structures in vertebrates and invertebrates. The Drosophila hh gene has now been shown to generate two predominant protein species that are derived by an internal autoproteolytic cleavage of a larger precursor. Mutations that reduced the efficiency of autoproteolysis in vitro diminished precursor cleavage in vivo and also impaired the signaling and patterning activities of the HH protein. The two HH protein species exhibited distinctive biochemical properties and tissue distribution, and these differences suggest a mechanism that could account for the long- and short-range signaling activities of HH in vivo.
Amino Acid Sequence↗
Island flap anoplasty for anal canal stenosis and mucosal ectropion.
Anoplasty is a technique used to treat patients with mucosal ectropion and anal canal stenosis. The island flap design has been demonstrated to have distinct advantages over the older forms of anoplasty. Few reports have addressed the long-term results of this procedure. A review of all patients undergoing island flap anoplasty between 1987 and 1992 was performed. Twenty-eight patients were identified. Indications for anoplasty included anal stenosis in 20 patients and mucosal ectropion in eight patients. Complications included five minor wound separations and one urinary tract infection with subsequent clostridium difficile enterocolitis. In-office follow-up averaged 7 months, at which time all patients were completely healed and significantly improved. Follow-up by phone was performed in 23 patients, extending their follow-up to 36 months. Based on the phone questionnaire, 91 per cent of the patients judged their symptoms as improved, and 9 per cent of patients judged their symptoms as unchanged. No patient judged their condition has worsened. Compared with the earlier forms of anoplasty, the island flap anoplasty is associated with a more simple design, less morbidity, and excellent long-term outcome. The island flap anoplasty should be the preferred design in cases of mucosal ectropion and anal stenosis.
Aged↗
Dependence of calmodulin localization in the retina on the NINAC unconventional myosin.
Calmodulin is a highly conserved regulatory protein found in all eukaryotic organisms which mediates a variety of calcium ion-dependent signalling pathways. In the Drosophila retina, calmodulin was concentrated in the photoreceptor cell microvillar structure, the rhabdomere, and was found in lower amounts in the sub-rhabdomeral cytoplasm. This calmodulin localization was dependent on the NINAC (neither inactivation nor afterpotential C) unconventional myosins. Mutant flies lacking the rhabdomere-specific p174 NINAC protein did not concentrate calmodulin in the rhabdomere, whereas flies lacking the sub-rhabdomeral p132 isoform had no detectable cytoplasmic calmodulin. Furthermore, a defect in vision resulted when calmodulin was not concentrated in the rhabdomeres, suggesting a role for calmodulin in the regulation of fly phototransduction. A general function of unconventional myosins may be to control the subcellular distribution of calmodulin.
Animals↗
Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis.
The Drosophila ninaC locus encodes a rhabdomere specific protein (p174) with linked protein kinase and myosin domains, required for a wild-type ERG and to prevent retinal degeneration. To investigate the role for linked kinase and myosin domains, we analyzed mutants generated by site-directed mutagenesis. Mutation of the kinase domain resulted in an ERG phenotype but no retinal degeneration. Deletion of the myosin domain caused a change in the subcellular distribution of p174 and resulted in both ERG and retinal degeneration phenotypes. Temperature-sensitive mutations in the myosin domain resulted in retinal degeneration, but no ERG phenotype. These results indicated that the ERG and retinal degeneration phenotypes were not strictly coupled suggesting that the myosin domain has multiple functions. We propose that the role of the kinase domain is to regulate other rhabdomeric proteins important in phototransduction and that the myosin domain has at least two roles: to traffic the kinase into the rhabdomeres and to maintain the rhabdomeres.
Alleles↗
Arterial thromboembolic complications of inflammatory bowel disease. Report of three cases.
Extraintestinal manifestations of inflammatory bowel disease are legion and are demonstrated in one-third of those afflicted. In general, they do not mandate surgery. Three patients with active pancolonic ulcerative colitis developed arterial thromboembolic complications prior to surgical treatment. Thromboembolic complications are not fully understood, as is evidenced by the paucity of information in the literature, and only sporadic cases of arterial thromboemboli are found. These have been described primarily in the postsurgical patient. To ascertain whether others have encountered similar cases, a survey form was distributed to members of The American Society of Colon and Rectal Surgeons, yielding an additional 54 patients with thromboembolic complications. Approximately two-thirds were deep venous thromboses and/or pulmonary emboli. Ten patients had cerebrovascular accidents, and eight had arterial emboli. Eleven patients, over 21 percent, suffered multiple events. There were four resultant mortalities. An arterial thromboembolic event in a patient with ulcerative colitis is usually associated with pancolonic disease, has a poor long-term prognosis, and is an indication for colectomy.
Adult↗
Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells.
The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.
Aging↗