Esophageal atresia and tracheoesophageal fistula associated with tetralogy of Fallot: a review of mortality.

INTRODUCTION: Oesophageal atresia ± tracheoesophageal fistula (EA/TEF) associated with congenital heart disease (CHD) carries a worse prognosis than EA/TEF alone. Though the Spitz classification takes major CHD into account, there are no data regarding survival with the specific combination of EA/TEF and Tetralogy of Fallot (TOF). With advances in postnatal care, we hypothesised that, survival is improving in these complex patients. This study reports morbidity and mortality outcomes of newborns with oesophageal atresia and TOF cardiac malformations METHODS: All patients with EA/TEF and TOF treated at Alder Hey Children's Hospital between the years 2000-2020, were identified. Data sets regarding gestation, birth weight, associated anomalies, operative intervention, morbidity, and mortality were analysed. RESULTS: Of a total of 350, EA/TEF patients 9 (2.6%) cases had EA/TEF associated with TOF (M:F 4:5). The median gestational age was 35/40 (range 28-41 weeks) with a median birth weight of 1790 g (range 1060-3350 g). Overall survival was 56% (5/9 cases) and all survivors remain under follow up (range 37-4458 days). Surgical strategies for managing EA/TEF with Fallot's tetralogy included 6/9 primary repairs and 3/9 cases with TEF ligation only (+ gastrostomy ± oesophagostomy). CONCLUSIONS: This study reports outcome data from one of the largest series of EA TEF patients with Fallot's tetralogy. Whilst outcomes may be challenging for this unique patient cohort, survival metrics provide important prognostic information that can be widely shared with health care teams and parents.

HBsAg(+) donor as a kidney transplantation deceased donor.

BACKGROUND: The organ shortage and high prevalence of hepatitis B (HB) infection in the general population are important issues in Taiwan. It is difficult for us to abandon HBsAg(+) donors. Hereby we present our experience transplanting kidneys from deceased donors with HB virus infection. METHODS: From November 1977 to March 2007, 21 patients with end-stage renal disease received kidney grafts from 12 HBsAg(+) deceased donors (3.92% of 306 donors). One of the 12 donors was hepatitis Be antigen (HBeAg) (+), and 5 displayed antibody to hepatitis core antigen (anti-HBc) (+). Four of the 21 recipients were HBsAg(+) before transplantation. RESULTS: Four HBsAg(+) recipients remained surface antigen positive after transplantation. One of them died of an intracranial hemorrhage. Two (11.76%) of the other 17 HBsAg(-) recipients became HBsAg(+), 1 of whom died of hepatic failure and the other of sepsis. The other 15 HBsAg(-) recipients (88.23%) remained HBsAg(-) after transplantation. They displayed normal serum levels of aspartate aminotransferase/alanine aminotransferase during the follow-up period. The 5-year patient and graft survivals were 85.15% and 61.14%, respectively. CONCLUSION: Although the number of patients is relatively small, it does suggest that a kidney allograft from an HBsAg(+) deceased donor transplanted to an HBsAg(+) or (-) recipient is safe. This strategy shortens the waiting time. Additional prophylactic HB immunoglobulin and antiviral medications are also suggested. Frequent surveillance after transplantation is essential.

Isodomoic acids A and C exhibit low KA receptor affinity and reduced in vitro potency relative to domoic acid in region CA1 of rat hippocampus.

Several natural isomers of the seizurogenic neurotoxin domoic acid (DA) have been found to occur at up to mg/kg levels in shellfish. The aim of the current study was to assess the neurotoxic potency of isodomoic acids A and C (Iso-A and Iso-C), recently isolated from commercial shellfish. Hippocampal slices were obtained from young adult rats and maintained in a tissue recording chamber. Synaptically evoked population spikes were recorded in region CA1 before and after exposure to DA or its isomers. Both Iso-A and Iso-C produced transient neuronal hyperexcitability followed by a dose-dependent suppression of population spikes, but were, respectively, 4- and 20-fold less potent than DA (spike area: EC50 DA=237 nM; Iso-A=939 nM; Iso-C=4.6 microM). In the hippocampus, DA preconditioning induces tolerance to subsequent DA toxicity. However, in the present study neither Iso-A nor Iso-C were effective as preconditioning agents. Competitive binding studies using homomeric GluR6 kainate (kainic acid, KA) receptors showed the affinity of Iso-A to be 40-fold lower than DA (Ki DA=3.35 nM; Iso-A=130 nM). Together with earlier work showing Iso-C affinity at GluR6 receptors to be 240-fold lower than DA, our results suggest that neuroexcitatory effects of Iso-A in CA1 may involve both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and KA receptors, while Iso-C likely involves the activation of AMPA receptors alone.

Hepatocellular carcinoma after renal transplantation: the long-term impact of cirrhosis on chronic hepatitis B virus infection.

Hepatocellular carcinoma (HCC) is the most common posttransplantation malignancy in hepatitis B virus (HBV) endemic areas. The aim of this study was to review the significant effect of liver cirrhosis on the outcome of renal allograft recipients with chronic hepatitis B. We performed a retrograde analysis of the clinical presentations of 66 hepatitis B surface antigen-positive kidney allograft recipients during the past 25 years with a mean follow-up of 76 months. Seven patients were diagnosed with HCC. The patients were subgrouped into cirrhotic versus noncirrhotic liver cohorts. Among renal allograft recipients with HBV infection, patients with cirrhotic livers had a higher risk of HCC (P = .003) and mortality (P = .025) than those with a noncirrhotic liver. The outcome was poor among the cirrhotic liver group. Pretransplantation liver biopsy may be indicated for the recipient candidate with HBV infection. Liver cirrhosis may be an exclusion criterion for the renal transplant waiting list due to the high incidence of HCC and the poor patient survival.

How to Improve the Positive Predictive Value of Urinary Decoy Cell Surveillance for Polyomavirus BK-Associated Nephropathy in Kidney Transplant Patients.

BACKGROUND: Polyomavirus BK-associated nephropathy (BKVN) has been a serious problem after kidney transplantation. Detection of urinary decoy cells (UDCs) and assessment of polyomavirus BK nucleic acids by polymerase chain reactions (PCRs) are currently used, noninvasive tests. PCRs have better positive predictive value (PPV) but higher cost and lower accessibility. This study investigated ways to improve the PPV of UDCs for BKVN prediction. METHODS: From 2000 to 2013, kidney transplant recipients with sustained UDCs for more than half a month and who had received allograft biopsies were enrolled. We analyzed the PPV of UDCs for BKVN with 2 variables: (i) the percentage changes in serum creatinine (SCr) levels and (ii) the duration of sustained UDCs by receiver operating characteristic (ROC) curve analysis; we predicted the percentage changes in SCr levels with the corresponding PPV using a linear regression model. RESULTS: BKVN was diagnosed in 26 of 68 enrolled patients. The percentage changes in SCr levels significantly deteriorated in the BKVN group during 1-2 months of UDC positivity. According to ROC curve analysis, percentage changes in SCr levels had a significant discriminating power for BKVN during 1-1.5 month, and if the percentage changes in SCr levels were >19%, the PPV of UDCs for BKVN was 50%. CONCLUSIONS: An UDC surveillance program is a judicious strategy to predict BKVN in kidney transplant patients, particularly when graft renal function shows deterioration after 1 month of UDC positivity.

Evidence for selective regulation of the phosphorylation of myocyte proteins by isoproterenol and prostaglandin E1.

Both isoproterenol and prostaglandin E1 increased the activation state of cyclic AMP-dependent protein kinase in cultured myocytes; however, only isoproterenol enhanced phosphorylase activity and contractile state. Following the incubation of intact myocytes with 32PO3-(4), 32 phosphoproteins were resolved from total cellular proteins by electrophoresis in sodium dodecyl sulfate polyacrylamide gels followed by autoradiography. Isoproterenol stimulated 32PO3-(4) incorporation into 16 proteins, including 2 phosphoproteins not observed under control conditions. By contrast, prostaglandin E1 neither caused a measurable change in the protein phosphorylation pattern nor interfered with isoproterenol's capacity to do so. Isoproterenol stimulated myocyte protein phosphorylation in either the presence or absence of extracellular Ca2+. The results suggest that the regulation of protein phosphorylation following adenylate cyclase stimulation is: (1) an agonist-specific process and not due solely to a random accumulation of intracellular cycle AMP and activation of protein kinase; (2) the Ca2+ mobilization component of beta-receptor activation does not account for the paradoxical effects of isoproterenol and prostaglandin E1; (3) activation of cyclic AMP-dependent protein kinase does not always result in an enhancement of protein phosphorylation.

Temporal inhibition of calmodulin in the nucleus.

Calmodulin (CaM) acts as a primary mediator of calcium signaling by interacting with target proteins. We have previously shown that nuclear CaM is critical for cell cycle progression using a transgene containing four repeats of a CaM inhibitor peptide and nuclear targeting signals (J. Wang et al., J. Biol. Chem. 270 (1995) 30245 30248; Biochim. Biophys. Acta 1313 (1996) 223-228). To evaluate the role of CaM in the nucleus specifically during S phase of the cell cycle, a motif which stabilizes the mRNA only during S phase was included in the transgene. The CaM inhibitor mRNA transcript contains a self-annealing stem-loop derived from histone H2B at the 3' end. This structure provides stability of the mRNA only during S phase, thereby restricting CaM inhibitor expression to S phase. The inhibitor accumulates in the nucleus, particularly in the nucleoli. Flow cytometric analysis demonstrated that the CaM inhibitor is expressed in S and G2. Transfected cells show growth inhibition and a reduction in DNA synthesis. The CaM inhibitor peptide is a versatile reagent that allows spatial as well as temporal dissection of calmodulin function.

28S ribosome degradation in lymphoid cell apoptosis: evidence for caspase and Bcl-2-dependent and -independent pathways.

Apoptosis, a physiological form of cell death, is characterized by the activation of a program that kills cells and recycles their constituents. We have used thymoma cell lines to examine the role of Bcl-2 and caspases in ribosomal destruction during apoptosis. Glucocorticoid- and calcium ionophore (A23187)-induced apoptosis of S49 Neo cells resulted in both 28S rRNA and DNA degradation. Interestingly, anisomycin, a potent protein synthesis inhibitor, also induced 28S rRNA and DNA fragmentation suggesting that the responsible nucleases are present in the viable cells and become activated during apoptosis. The anti-apoptotic protein, Bcl-2, inhibited both glucocorticoid- and anisomycin-induced DNA and 28S rRNA degradation but could not protect against A23187-induced nucleic acid degradation. We next examined the role of caspase activation in the generation of 28S rRNA degradation through the use of ZVAD, a general caspase inhibitor. Under conditions where ZVAD substantially decreased 28S rRNA degradation induced by glucocorticoid or anisomycin, no decrease was observed when A23187 was used to induce apoptosis. Surprisingly, RNA degradation, like DNA degradation, occurs exclusively in shrunken lymphocytes but not those with normal cell volume despite equivalent exposure of the cells to the apoptotic signals. Together, these findings indicate the ribosome is a specific target for death effectors during apoptosis and that a caspase/Bcl-2-independent pathway exists to activate its destruction.

Crystallization of the motile major sperm protein (MSP) of the nematode Ascaris suum.

We have obtained orthorhombic crystals (space group P2(1)2(1)2(1), a = 72 A, b = 78 A, c = 452 A) of the major cytoskeletal protein associated with the amoeboid motility of Ascaris sperm. These crystals diffract past 3.5 A and appear to be constructed from arrays of helical fibres of the major sperm protein. The fibres within the crystals appear to be closely analogous to those seen in vitro and in vivo.

The motile major sperm protein (MSP) of Ascaris suum forms filaments constructed from two helical subfilaments.

The amoeboid motility nematode sperm is mediated by cytoskeletal filaments composed of major sperm protein (MSP). We have used electron microscopy and image processing to show that MSP filaments are constructed from two subfilament strands which are themselves formed from a helical arrangement of subunits. The subfilaments are based on left-handed helices of pitch 9 nm that then coil along right-handed helical tracks of pitch 22.5 nm to form filaments. The subfilaments appear to be indistinguishable from the helices present in orthorhombic crystals of MSP. Because in filaments the subfilaments are themselves helical, not all subunits are able to participate in protein-protein interactions between different strands. One consequence of this interaction geometry is that the same molecular interactions that function to assemble subfilaments into filaments can also be used to assemble filaments into larger supramolecular assemblies such as the macrofibres formed in vitro and the fibre bundles found in vivo in sperm pseudopods. These results indicate the importance of filament bundling in addition to vectorial filament assembly in amoeboid cell motility.

Expression of a parathyroid hormone-like protein and its receptor during differentiation of embryonal carcinoma cells.

Differentiation of mouse embryonal carcinoma cells to the parietal endoderm phenotype is associated with expression of PTH-responsive adenylate cyclase. A PTH-like protein (PLP), which binds to PTH receptors and activates adenylate cyclase in classical PTH target cells was recently isolated and cloned. We assessed whether the parietal endoderm phenotype is associated with the expression of PLP or its receptor. A 1.4-kilobase PLP transcript was detected in the mouse parietal endoderm cell line PYS-2. No hybridizing transcripts were evident in undifferentiated mouse embryonal carcinoma cells PSA-1 or F9. However, differentiation of these cells to parietal endoderm, either spontaneously (PSA-1) or by treatment with retinoic acid and dibutyryl cAMP (F9), resulted in expression of the 1.4-kilobase PLP message. Undifferentiated F9 cells displayed negligible specific binding of [125I]PLP-(1-34)amide. When F9 cells were induced to differentiate to parietal endoderm, specific binding sites for [125I]PLP-(1-34)amide were expressed in parallel with PLP-responsive adenylate cyclase. These receptors, like those in classical PTH target tissues, displayed identical affinity (Kd = 5.2 nM) for bPTH-(1-34) and hPLP-(1-34)amide; with binding capacity (Bmax) of 6.6 x 10(4) sites/cell. In the presence of retinoic acid, exogenous PLP substituted for dibutyryl cAMP in a concentration-dependent fashion in promoting the differentiation of F9 cells to parietal endoderm. Thus, both PLP mRNA and PLP receptors coupled to adenylate cyclase are expressed during the differentiation of mouse embryonal carcinoma cells. Increased cAMP levels produced by autocrine stimulation of PLP receptors by PLP may contribute to differentiation of embryonal carcinoma cells into parietal endoderm.

G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes.

Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10(-4) M) or guanosine 5'-0-(3-thiotriphosphate, GTP gamma S, 10(-5) M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTP gamma S produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H]IP3, respectively. The stimulatory effects of GTP and GTP gamma S were dose dependent (GTP ED50 = 3.9 x 10(-6) M; GTP gamma S ED50 = 2.5 x 10(-7) M) and progressive over 10 minutes and required the presence of Mg2+.GTP (10(-4) M) and GTP gamma S (10(-5) M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDP beta S) alone did not alter [3H]IP2 or [3H]IP3 production but at 10(-4) M blocks the stimulatory effects of GTP and GTP gamma S. NaF (3 x 10(-2)M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of kistrin on bone resorption in vitro and serum calcium in vivo.

In many cell systems, cell-cell and cell-matrix interactions are mediated by integrins, a family of cell surface heterodimeric glycoprotein receptors. Osteoclast integrins may play a role in the process of bone resorption. Osteoclasts express the alpha v and beta 3 subunits of the vitronectin receptor and adhere to a wide range of proteins in vitro, all which contain the amino acid sequence Arg-Gly-Asp (RGD), an adhesion site recognition sequence common to many protein ligands that bind to integrins. The effect of kistrin, an RGD-containing snake venom protein, on osteoclast-mediated bone resorption was investigated in vivo and in vitro. When kistrin was infused into normocalcemic and hypercalcemic mice, serum calcium was significantly lowered at 3 and 6 h after the start of infusion, indicating an inhibitory effect on osteoclast activity in vivo. In vitro, kistrin potently inhibited bone resorption by isolated rat osteoclasts cultured on slices of bovine bone, and kistrin also inhibited the attachment of 293 cells expressing recombinant human alpha v beta 3 to fibrinogen (IC50 = 1 nM). These results indicate the potential therapeutic use of RGD-containing molecules for hypercalcemia of malignancy or for other disorders associated with bone loss.

Binding and cross-linking of 32P-labeled human relaxin to human uterine cells and primary rat atrial cardiomyocytes.

Relaxin is known for its function in parturition and has been suggested to participate in the regulation of blood pressure, heart rate, and the release of neuropeptides such as oxytocin and vasopressin. Consistent with the physiological roles of relaxin, high affinity relaxin receptors have been demonstrated in the rat uterus, brain, and cardiac atrium. Here we report the binding and cross-linking of a biologically active, 32P-labeled human relaxin to a human uterine cell line and primary rat atrial cardiomyocytes. Relaxin binding to the human uterine cells consisted of a single class of high affinity sites (Kd = approximately 0.44 nM) with approximately 1082 +/- 62 binding sites/cell. Binding and cross-linking of relaxin to the human uterine cells and rat atrial cardiomyocytes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the putative relaxin receptor showed a major component with an apparent M(r) greater than 220 kilodaltons and a minor component of approximately 36 kilodaltons, and was not disulfide linked. The binding and cross-linking of [32P]relaxin could be displaced by unlabeled relaxin in a concentration-dependent manner, but not by a 1000-fold molar excess of insulin, insulin-like growth factor I (IGF-I), or IGF-II. These data suggested that the relaxin receptor was similar in size but distinct from the insulin, IGF-I, and IGF-II receptors.

Thrombin stimulates inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells.

Human alpha-thrombin is known to elicit bone resorption in vitro and has been proposed as a mediator of increased bone turnover in inflammatory diseases. We used UMR 106-H5 rat osteoblast-like osteosarcoma cells to explore the signal transduction mechanism utilized by thrombin in bone. Thrombin produced a dose-dependent increase in the accumulation of [3H]inositol phosphates (IPs) in UMR 106-H5 cells prelabeled with [3H]myo-inositol (EC50 15 U/ml). In saponin-permeabilized cells, GTP gamma S increased [3H]IP production, whereas GDP beta S inhibited the response to both GTP gamma S and thrombin, indicating involvement of a G-protein in thrombin action. Thrombin produced a dose-dependent increase in intracellular free calcium (Cai2+) in UMR 106-H5 cells (EC50 1 U/ml; maximal increase 4-fold), as well as a small (20%) increase in [3H]thymidine incorporation. Treatment of UMR 106-H5 membranes with pertussis toxin (PT) and [32P]NAD+ resulted in labeling of a 40-kDa protein. However, pretreatment of cells with a dose of PT sufficient to produce maximal endogenous labeling of this protein failed to influence thrombin action on IP accumulation, Cai2+, or [3H]thymidine incorporation. In contrast, PT treatment of CCL39 hamster lung fibroblasts significantly blunted thrombin-stimulated [3H]IP accumulation and [3H]thymidine incorporation. These results suggest that thrombin raises Cai2+ in UMR 106-H5 cells by activating polyphosphoinositide-specific phospholipase C. Whereas in fibroblasts and platelets, thrombin receptors appear to couple to both PT-sensitive and PT-insensitive G-proteins, only a PT-insensitive G-protein appears to mediate thrombin action in UMR 106-H5 cells. Either these cells lack the relevant PT-sensitive G-protein or they possess thrombin receptors that selectively couple to a pertussis toxin-insensitive G-protein.

Thrombin and parathyroid hormone mobilize intracellular calcium in rat osteosarcoma cells by distinct pathways.

The mechanisms by which PTH and thrombin mobilize intracellular Ca2+ (Cai2+) were examined in UMR 106-H5 rat osteosarcoma cells. Bovine PTH-(1-34) (24 pM to 240 nM) produced a dose-dependent increase in Cai2+ (EC50, 3 nM), which returned to baseline within 75 sec. Human alpha-thrombin produced an increase in Cai2+ (ECmax, 10 U/ml) which was similar to that of PTH with respect to both magnitude and time course. Chelation of extracellular calcium with 5.0 mM EGTA did not alter the Cai2+ response to either PTH or thrombin. When added together at maximally effective concentrations, PTH and thrombin produced additive effects on Cai2+ in the presence and absence of EGTA. The additive effects of PTH and thrombin on Cai2+ were confirmed at the single cell level, using laser-based image analysis. Bradykinin (1 microM) produced a significant increase in Cai2+ in UMR 106-H5 cells which was of lesser magnitude than the peak 2- to 3-fold increase elicited by PTH or thrombin. Preexposure of cells to 10 U/ml thrombin for 2 min abolished the Cai2+ response to bradykinin, whereas preexposure to 240 nM PTH had no effect on the Cai2+ response to bradykinin. Thrombin elicited a rapid increase in the accumulation of 3H-labeled inositol phosphates (IP2 and IP3) in UMR 106-H5 cells, with increases in [3H]1,4,5-IP3 detectable as early as 15 sec after the addition of thrombin. Bradykinin increased [3H]IP production to a lesser extent than thrombin, whereas PTH neither increased [3H]IP accumulation nor potentiated the [3H]IP response to thrombin. The results suggest that thrombin and bradykinin mobilize Cai2+ from a shared IP3-responsive calcium pool, whereas PTH may use signals in addition to 1,4,5-IP3 to mobilize calcium from a distinct cellular calcium pool. Alternatively, specific calcium compartmentalization exists, and there is differential coupling of these agonists to the 1,4,5-IP3/Cai2+ pathway.

Biological response to ErbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression.

The human epidermal growth factor receptor (HER or ErbB) family consists of four distinct members, including the epidermal growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors plays an important role in the regulation of cell proliferation, differentiation, and survival in several different tissues. Binding of a specific ligand to one of the ErbB receptors triggers the formation of specific receptor homo- and heterodimers, with ErbB2 being the preferred signaling partner. We analyzed the levels of various ErbB receptor messenger RNAs in a series of nontransformed cell lines by real time quantitative RT-PCR. The cell lines chosen were derived from a variety of tissues, including pancreas, lung, heart, and nervous system. Further, we measured biological responses in these cell lines upon treatment with EGF, betacellulin, and two types of neuregulins, heregulin and sensory and motor neuron-derived factor. All cell lines examined expressed detectable levels of ErbB2. High levels of expression of ErbB3 were correlated with responsiveness to heregulin and sensory and motor neuron-derived factor, whereas high levels of EGFR expression were correlated with responsiveness to EGF and betacellulin. Moreover, the sensitivity of a cell line to ErbB ligands was also correlated with the levels of expression of the appropriate ErbB receptors in that cell line. These results are consistent with our hypothesis that appropriate biological responsiveness to ErbB ligands is determined by the levels of expression of specific ErbB receptor combinations within a given tissue.

Growth hormone stimulates the formation of sn-1,2-diacylglycerol in rat hepatocytes.

The transmembrane signaling events of GH were investigated in the liver, a major target organ of GH action. Recombinant human GH when added to freshly isolated rat hepatocytes rapidly stimulated the production of sn-1,2-diacylglycerol (DAG). The generation of DAG was biphasic with the first transient peak observed at 2 min and the second peak at 15 min (1.2-fold and 1.4-fold over control, respectively). Levels of DAG continued to be elevated above those in control cells at 30 min. The response was dose-dependent with an EC50 of 0.15 nM. Both bovine GH and rat GH, which bind to the rat GH receptor but not to the PRL receptor, also stimulated DAG production. Similarly, human PRL, which binds to the PRL but not GH receptor, stimulated DAG formation to a comparable extent. These results suggest that production of DAG may be an early signaling event mediated by hormone stimulation of both the GH and PRL receptors.

Structural properties of the renal parathyroid hormone receptor: hydrodynamic analysis and protease sensitivity.

The highly specific ligand [125I]bovine (b) PTH-(1-34) and a chemical cross-linking technique were used to explore structural features of the canine renal cortical PTH receptor. Membranes isolated under conditions designed to inhibit endogenous proteolysis displayed a major 85K labeled PTH receptor moiety on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linked receptors were solubilized with Lubrol-PX and partially purified by affinity chromatography on wheat germ agglutinin-agarose, and their hydrodynamic properties were assessed [Stokes radius = 7.3 +/- 0.1 nm; sedimentation coefficient = 6.4 +/- 0.2S; partial specific volume = 0.758 +/- 0.01 ml/g; frictional coefficient = 1.68 +/- 0.04; mol wt (Mr) = 216,000 +/- 14,000]. Corrections for detergent binding and for the presence of carbohydrate yielded an estimated Mr of 166,000 +/- 11,000 for the solubilized PTH receptor. Thus, the renal PTH receptor is oligomeric, with a Mr approximating that expected of a homodimer of 85K subunits. Peptide-mapping experiments revealed the presence within the 85K PTH receptor subunit of at least two major regions sensitive to proteolytic attack. Both elastase and an endogenous renal protease(s) cleaved the PTH receptor to a 70K form that is fully functional with respect to high affinity, guanyl nucleotide-sensitive PTH binding. Cleavage in a second domain by elastase, S. aureus V8 protease, or chymotrypsin generated a 50K labeled PTH receptor fragment. Cleavage at this second site was prevented by prior occupancy of the receptor with [125I]bPTH-(1-34), suggesting that this domain may be functionally important. Reduction of receptor disulfide bonds with dithiothreitol and beta-mercaptoethanol released a low Mr (less than or equal to 14K) labeled PTH receptor component, similar treatment of renal membranes abolished specific PTH binding, indicating that an intact disulfide bond(s) is essential for receptor function. These results provide new insights into the structural basis of PTH receptor function.

Age-dependent increase of mitochondrial DNA deletions together with lipid peroxides and superoxide dismutase in human liver mitochondria.

We previously reported an age-dependent deterioration of mitochondrial respiration as well as two age-associated mitochondrial DNA (mtDNA) deletions in the human liver. In this study, we further determined the relative quantities of the deleted mtDNAs in liver biopsies from 64 subjects of different ages. The results showed that both mtDNA deletions increase in frequency and quantity with age. Moreover, we measured hepatic lipid peroxides (malondialdehyde; MDA) of isolated mitochondria and manganese superoxide dismutase (Mn-SOD) activity of submitochondrial particles. We found a significant age-dependent increase in both MDA and Mn-SOD levels in liver mitochondria. These results confirm the previous contention that enhanced generation of lipid peroxides in the mitochondria during the aging process may damage mtDNA, and mtDNA deletions may be one of the important factors contributing to aging in humans.