Laparoscopic Roux-en-Y gastric bypass in a morbidly obese patient with renal transplant: a case report.

Renal transplant is the only curative treatment for end-stage renal disease. As diabetes and obesity are the major causes of graft failure and post-transplant complication, it is important to manage obesity in patients with renal transplant. Herein, we report a case of a morbidly obese renal-transplant patient with poorly controlled diabetes who received bariatric surgery. A 34-year-old obese Taiwanese man with type 2 diabetes had end-stage renal disease that had progressed since 2008, when he had commenced hemodialysis (January 2008) and had a renal transplant (July 2008). Because of persistent obesity and poorly controlled diabetes, he received LRYGB at Chiayi Christian hospital on 18 August 2010. In the month that followed, he lost 10 kg. His serum creatinine decreased to 1.11 mg/dL (1.4 mg/dL, preoperative) and his hemoglobin A1c decreased to 8.5% (10.4%, preoperative). These results indicate that, in obese renal transplant patients, LRYGB may be employed to treat obesity, control diabetes and stabilize or improve the renal function.

Polymorphism of transmembrane region of MICA gene and Kawasaki disease.

Kawasaki disease is a febrile disease of children complicated with vasculitis of the coronary arteries and potential aneurysm formation. It has been recognized worldwide and appears to be increasing in frequency. Studies have found that Kawasaki disease is associated with major histocompatibility complex (MHC) class I B antigens. The MHC-class-I-chain-related gene A (MICA) is located near HLA-B. It has a triplet repeat microsatellite polymorphism in the transmembrane region. We investigated the microsatellite polymorphism in children with Kawasaki disease and controls. Seventy children (46 boys), age at diagnosis 1.68 +/- 1.69 years, with Kawasaki who were treated with aspirin as well as intravenous gamma-globulin were enrolled. Control subjects consisted of 154 children (87 boys), age 2.81 +/- 2.12 years. Phenotype frequency of allele A4 in patients with aneurysm formation was significantly lower than in patients without aneurysms [relative risk (RR) = 0.06, 95% confidence interval (CI) = 0.01-0.48, p = 0.00469, pc = 0.0232] and showed a similar tendency when compared with controls. Gene frequency of allele A4 was also significantly lower in patients who developed aneurysms than in patients who did not (RR = 0.07, 95% CI = 0.01-0.57, p = 0.0057, pc = 0.0282). Gene frequency of allele A5 showed a tendency to be higher in patients who developed aneurysms than in controls (RR = 2.35, 95% CI = 0.98-5.63, p = 0.0486, pc = 0. 220). Allele A5.1 tended to be negatively associated with Kawasaki disease (RR = 0.57, 95% CI = 0.35-0.93, p = 0.022, pc = 0.105). Our study showed that allele A4 was negatively associated with coronary aneurysm formation in Kawasaki disease. This suggests that allele A4 protects the children with Kawasaki disease from developing coronary aneurysms after aspirin and gamma globulin therapy.

The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate.

Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

Mammalian phospholipase D structure and regulation.

The recent identification of cDNA clones for phospholipase D1 and 2 has opened the door to new studies on its structure and regulation. PLD activity is encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiple specialized steps in receptor dependent and constitutive processes of secretion, endocytosis, and membrane biogenesis.

Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway.

The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulin-dependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles.

Structural analysis of human phospholipase D1.

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been proposed to play roles in numerous cellular pathways including signal transduction and membrane vesicular trafficking. We previously reported the cloning of two mammalian genes, PLD1 and PLD2, that encode PLD activities. We additionally reported that PLD1 is activated in a synergistic manner by protein kinase c-alpha (PKC-alpha), ADP-ribosylation factor 1 (ARF1), and Rho family members. We describe here molecular analysis of PLD1 using a combination of domain deletion and mutagenesis. We show that the amino-terminal 325 amino acids are required for PKC-alpha activation of PLD1 but not for activation by ARF1 and RhoA. This region does not contain the sole PKC-alpha interaction site and additionally functions to inhibit basal PLD activity in vivo. Second, a region of sequence unique to PLD1 (as compared with other PLDs) known as the "loop" region had been proposed to serve as an effector regulatory region but is shown here only to mediate inhibition of PLD1. Finally, we show that modification of the amino terminus, but not of the carboxyl terminus, is compatible with PLD enzymatic function and propose a simple model for PLD activation.

Calcinosis cutis following extravasation of calcium gluconate in neonates.

Neonatal hypocalcemia is not an uncommon condition, especially in the premature neonate. It is effectively treated by intravenous infusion with calcium gluconate. We treated nine neonates with subcutaneous calcium deposition following calcium replacement with calcium gluconate from Jan. 1997 to Dec. 1997. Three of the infants were born to diabetic mothers, two had perinatal asphyxia and four were born prematurity. The average dosing number was 7.4 (5 to 9 doses). The onset of calcinosis cutis was 5 to 11 days after the first dose. The replacement of calcium gluconate caused amorphous masses at the site of extravasation and contracture of joint movement. A radiographic study was performed to determine the extent and course of extravasation, and areas remote from the infusion site also showed calcification. There is no specific mode of treatment except supportive management and a skin graft. The patient could functionally recover with cosmetic residue. In our follow-up clinics, all infants completely recovered without functional limitations.

Endophthalmitis as a complication of meningococcal meningitis: report of one case.

Metastatic meningococcal endophthalmitis, although rare, is a rapidly progressive and sight-threatening infection. We present a 10-month-old infant with meningococcal meningitis who developed unilateral metastatic endophthalmitis. If patients develop a sepsis-like picture with cloudy cornea and purulent conjunctivitis, we have to consider the possibility of endophthalmitis and full ophthalmological evaluations are indicated. Treatment should be started as early as possible. The outcome of endophthalmitis is frequently permanent visual impairment. Endophthalmitis is a true medical emergency requiring early antibiotic therapy with full dose of antimicrobials to avoid morbidity and blindness.

Aneurysm of the ascending aorta in a neonate.

Aneurysms of the thoracic aorta rarely occur in children. We present a female neonate who was referred to our hospital due to a heart murmur associated with cough and fever at 22 days of age. Both the echocardiography and aortography displayed an aneurysm of the ascending aorta at the aortic root. A patent ductus arteriosus (PDA) flow was detected on admission but it was not detectable when she was 3 months old. Neither physical characteristics of Marfan nor Turner syndrome were found, but she has had a huge cutaneous hemangioma over the right trunk since birth. The aneurysm did not progress during one year of follow-up. The etiology might be idiopathic or medial agenesis. Surgery will be warranted only if the aneurysm enlarges.

Hydrops fetalis with complete heart block secondary to congenital lupus: report of one case.

Neonatal lupus erythematosus is an uncommon syndrome characterized by a congenital heart block and/or cutaneous lesion. We report a male newborn with neonatal lupus erythematosus presenting with complete heart block, cutaneous lesions, and hydrops. Transplacental passage of anti-SSA/Ro and anti-SSB/La antibodies were positive. Under the regimens of steroid for maternal systemic lupus erythematosus, perinatal anticongestive agents, and postnatal ventricular pacing with inotropic therapy, the infant died at the age of two days. The prognosis of hydrops fetalis secondary to neonatal lupus with complete heart block is usually fatal.

Molecular analysis of mammalian phospholipase D2.

The mammalian phosphatidylcholine-specific phospholipase D (PLD) enzymes PLD1 and PLD2 have been proposed to play roles in signal transduction and membrane vesicular trafficking in distinct subcellular compartments. PLD1 is activated in a synergistic manner in vitro by protein kinase C-alpha, ADP-ribosylation factor 1 (ARF1), and Rho family members. In contrast, PLD2 is constitutively active in vitro. We describe here molecular analysis of PLD2. We show that the NH2-terminal 308 amino acids are required for PLD2's characteristic high basal activity. Unexpectedly, PLD2 lacking this region becomes highly responsive to ARF proteins and displays a modest preference for activation by ARF5. Chimeric analysis of PLD1 and PLD2 suggests that the ARF-responsive region is in the PLD carboxyl terminus. We also inserted into PLD2 a region of sequence unique to PLD1 known as the "loop" region, which had been proposed initially to mediate effector stimulation but that subsequently was shown instead to be required in part for the very low basal activity characteristic of PLD1. The insertion decreased PLD2 activity, consistent with the latter finding. Finally, we show that the critical role undertaken by the conserved carboxyl terminus is unlikely to involve promoting PLD association with membrane surfaces.

Holocord intramedullary spinal cord astrocytoma: report of one case.

Intramedullary spinal cord astrocytoma in infants is relatively uncommon. Its occurrence is usually confined to the cervical and cervicothoracic regions. In this paper, we report on the case of a 4-month old male infant with low grade holocord intramedullary spinal cord astrocytoma. He had developed progressive weakness of the lower extremities over a month period. Neurological examination revealed flaccid paraplegia as well as complete loss of all modalities of sensation below the T10 level. MRI revealed a large intramedullary mass which was found to be an intramedullary astrocytoma at surgery. This case report presents the clinical features, radiographic findings, and treatment and outcome for this patient together with a review of relevant literature.

Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity.

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.

BACKGROUND: Activation of phospholipase D (PLD) is an important but poorly understood component of receptor-mediated signal transduction responses and regulated secretion. We recently reported the cloning of the human gene encoding PLD1; this enzyme has low basal activity and is activated by protein kinase C and the small GTP-binding proteins, ADP-ribosylation factor (ARF), Rho, Rac and Cdc42. Biochemical and cell biological studies suggest, however, that additional and distinct PLD activities exist in cells, so a search was carried out for novel mammalian genes related to PLD1. RESULTS: We have cloned the gene for a second PLD family member and characterized the protein product, which appears to be regulated differently from PLD1: PLD2 is constitutively active and may be modulated in vivo by inhibition. Unexpectedly, PLD2 localizes primarily to the plasma membrane, in contrast to PLD1 which localizes solely to peri-nuclear regions (the endoplasmic reticulum, Golgi apparatus and late endosomes), where PLD activity has been shown to promote ARF-mediated coated-vesicle formation. PLD2 provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis. CONCLUSIONS: PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2.

Different domains of mammalian ADP-ribosylation factor 1 mediate interaction with selected target proteins.

Mammalian ADP-ribosylation factor 1 (mARF1) is a small GTP-binding protein that is activated by a Golgi guanine nucleotide exchange factor. Once bound to the Golgi membranes in the GTP form, mARF1 initiates the recruitment of the adaptor protein 1 (AP-1) complex and coatomer (COPI) onto these membranes and activates phospholipase D1 (PLD1). To map the domains of mARF1 that are important for these activities, we constructed chimeras between mARF1 and Saccharomyces cerevisiae ARF2, which functions poorly in all of these processes except COPI recruitment. The carboxyl half of mARF1 (amino acids 95-181) was essential for activation by the Golgi guanine nucleotide exchange factor, whereas a separate domain (residues 35-94) was required to effectively activate PLD1 and to promote efficient AP-1 recruitment. Since residues 35-94 of mARF1 are critical for optimal activity in both PLD1 activation and AP-1 recruitment, we hypothesize that this region of ARF contains residues that interact with effector molecules.

Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family.

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

In cultured canine tracheal smooth muscle cells (TSMCs), muscarinic receptor stimulation led to phosphoinositide (PI) hydrolysis, formation of inositol phosphates (IPs), and mobilization of intracellular Ca2+. Desensitization of IPs accumulation and Ca2+ mobilization evoked by carbachol was investigated using [3H]inositol labelling and Ca(2+)-sensitive dye fura-2. Treatment of TSMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min blocked the carbachol-stimulated formation of IPs and mobilization of Ca2+. The concentrations of PMA that gave half-maximal and maximal inhibition of carbachol-induced IPs accumulation were 70 nM and 1 microM, respectively. The inhibitory effect of PMA on carbachol-induced responses was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. Treatment of TSMCs with PMA for 24 h, the cells remained the ability to response to carbachol-induced IPs accumulation and Ca2+ mobilization with the same extent as that observed in the control group. Inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit the responses. The KD and Bmax of the muscarinic receptor for [3H]N-methyl scopolamine binding were not significantly changed by PMA treatment for either 30 min or 24 h. The locus of this inhibition was further investigated by examining the effect of PMA on AlF4(-)-stimulated IPs accumulation in canine TSMCs. AlF4(-)-induced response was inhibited by PMA treatment, supporting that G protein(s) can be directly activated by AlF4- which was uncoupled to phospholipase C (PLC) by PMA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization of muscarinic receptors in neonatal rat cardiomyocytes.

[N-methyl-3H]scopolamine methylchloride ([3H]NMS) was used to characterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes. The specific binding of [3H]NMS was proportional to cell concentration, saturable with respect to [3H]NMS concentration, and time dependent. Scatchard analysis of binding isotherms showed that [3H]NMS bound to the freshly isolated and cultured cardiomyocytes with dissociation constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum receptor densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respectively. Heterogeneity of mAChRs was demonstrated by competitive binding experiments against [3H]NMS with M2 and M3 antagonists. These receptors (80%) exhibited high affinities for 11-([2-[(diethylamino)methyl]-1-piperidinyl]-acetyl)-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine similar to those of M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors and possessed high affinity for 4-diphenylacetoxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the basis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic monophosphate (cAMP) inhibition with high affinity (pKB 7.4), while it antagonized inositol phosphate formation with low affinity (pKB 6.5). 4-DAMP possessed high affinity in blocking inositol phosphate formation (pKB 9.0) and low affinity for antagonism of cAMP inhibition (pKB 7.7). Although no other muscarinic receptor mRNA has been detected in these cells, these data suggest the presence of a second population of mAChRs, which may not be identical to the classical cardiac "M2" receptors.