Biomedical subjects
Publications and source records attributed to L Kushner.
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BACKGROUND AND PURPOSE: We previously demonstrated that obstructed ureteropelvic junction (UPJ) segments from patients who had secondary pyeloplasty after endopyelotomy failure expressed transforming growth factor-beta1 (TGF-beta1) at levels significantly lower than patients who had primary pyeloplasty. In order to determine whether these differences in secreted TGF-beta1 are detectable preoperatively in the urine, the TGF-beta1 concentration of urine from patients undergoing endopyelotomy was determined and compared with that from subjects without urologic disease. MATERIALS AND METHODS: Bladder and renal pelvic urine from the obstructed side was obtained from patients (N = 34) undergoing primary endopyelotomy for UPJ obstruction. Bladder urine was also obtained from sex- and age-matched patients (N = 26) having no evidence of urinary tract obstruction. The TGF-beta1 concentration was determined by ELISA and normalized to the creatinine concentration. RESULTS: The bladder urine TGF-beta1 concentration was significantly (P < 0.02) higher in patients with UPJ obstruction (86.1+/-20.5 pg/mg of creatinine) than in those without obstruction (29.7+/-8.0 pg/mg creatinine). The TGF-beta1 concentration in the bladder urine of patients who underwent endopyelotomy and later returned because of UPJ obstruction (25.7+/-12.3 pg/mg of creatinine; N = 6) was not significantly different from the value in unobstructed patients but was significantly lower (P < 0.01) than in patients for whom endopyelotomy was successful (100+/-24.29 pg/mg of creatinine; N = 28). The renal pelvic urinary TGF-beta1 concentration was higher in patients for whom endopyelotomy was successful (772+/-490.1 pg/mg of creatinine) than in patients who underwent endopyelotomy and later returned because of UPJ obstruction (126.1+/-41.9 pg/mg of creatinine). CONCLUSIONS: These data suggest that preoperative concentration of TGF-beta1 in the bladder urine of patients with UPJ obstruction who fail endopyelotomy is not significantly different from that in subjects with no urologic disease and significantly lower than in those patients for whom endopyelotomy is successful. Thus, the preoperative bladder urine concentration of TGF-beta1 may assist in selecting patients for this operation, although further investigation is necessary.
The universe is not designed by a deity outside, beyond, and other than the universe. The design of the universe is experienced in its unity, dynamic interdependency, and interconnectedness. The designer is to be found in the experience of such design, not beyond it. Within Judaism, the Kabbalah is one expression of this religious vision of the universe.
BACKGROUND AND OBJECTIVE: Endopyelotomy relies on Davis' intubated ureterotomy principle of healing by secondary intention and smooth-muscle regeneration. Approximately 15% of endopyelotomies fail, and the restrictured segment almost always shows evidence of reactive fibrosis with little smooth-muscle regeneration. Previous data suggests that an elevation of TGF beta in obstructed ureteropelvic junctures may be necessary for successful tissue repair following endopyelotomy. The role of crossing vessels in endopyelotomy failure is very controversial. To better understand the pathophysiology of endopyelotomy failure, the expression of transforming growth factor-beta (TBG beta) in patients with a failed endopyelotomy and crossing vessels was compared with that in patients without crossing vessels, as well as those having primary pyeloplasty or a normal ureteropelvic junction (UPJ). MATERIALS AND METHODS: The expression of TGF beta was detected immunohistochemically in slide-mounted thin sections (4 microns) cut from paraffin-blocked adult UPJ segments obtained during primary pyeloplasty (N = 11), secondary pyeloplasty after failed endopyelotomy with documented crossing vessels (N = 10), secondary pyeloplasty after failed endopyelotomy without crossing vessels (N = 11), and normal UPJs removed during nephrectomy for purposes unrelated to obstruction (N = 11). Expression was graded on a scale of 0 to 4. RESULTS: The combined failed endopyelotomy group had a significantly (P < 0.05) lower level of TGF beta (1.9 +/- 0.7) than did primary obstructed UPJs (2.6 +/- 0.7). The TGF beta level in the crossing vessels group (1.9 +/- 0.7) did not differ from that in the group without crossing vessels (1.8 +/- 0.7), nor did it differ from that in the group with normal UPJs (1.6 +/- 0.7). As expected, primary obstructed UPJs had a significantly higher level of TGF beta than normal ones (P < 0.02). CONCLUSIONS: Obstructed UPJs that had failed endopyelotomy had a similarly reduced level of TGF beta whether or not crossing vessels were present. These data suggest that an elevation of TGF beta in obstructed UPJs may be necessary for successful tissue repair after endopyelotomy and that the presence of crossing vessels is probably not relevant.
PURPOSE: In acute pyelonephritis renal scarring may be decreased by immediate antibiotic therapy. Unfortunately in children there is often a delay in starting treatment, which increases the likelihood of renal scarring. In rodents immediate antibiotic therapy is effective for preventing renal scar formation resulting from experimentally induced pyelonephritis. However, the same treatment beginning 72 hours after infection does not prevent renal scarring in this paradigm. We examined whether delayed administration of the nonsteroidal anti-inflammatory agent ibuprofen only or combined with antibiotics suppresses renal scarring in a model of ascending pyelonephritis in rats. MATERIALS AND METHODS: An inoculum of 5x10(9) organisms per ml. of Escherichia coli strain BH-5 was instilled into the bladder of rats and the urethra was occluded for 4 hours. Groups of animals were and were not treated with 15 mg./kg. cefadroxil or 10 mg./kg. ibuprofen given twice daily for 5 days, or the 2 drugs combined. Treatment began 72 hours after inoculation. In an additional group of rats sterile phosphate buffered saline was instilled into the bladder. In each rat the kidneys were examined grossly and microscopically 6 weeks later. RESULTS: Combined antibiotics and ibuprofen significantly inhibited gross renal scarring compared with no treatment or antibiotics only (p<0.05). No difference in renal scarring was detected in animals that received no treatment versus those that received antibiotics or ibuprofen only (p>0.05). CONCLUSIONS: Renal scarring resulting from acute pyelonephritis in this rat model is not decreased by delayed treatment with antibiotics only. The addition of ibuprofen to antibiotic therapy is effective for decreasing renal scarring due to acute pyelonephritis even when treatment is delayed for 72 hours.
PURPOSE: Approximately 15% of patients with ureteropelvic junction obstruction have endopyelotomy failure and require an additional surgical procedure to remove the obstruction. Transforming growth factor-beta (TGF-beta), a cytokine which stimulates mesenchymal cell proliferation and extracellular matrix deposition, increases in the renal pelvis in response to obstruction. However, TGF-beta also is implicated in smooth muscle regeneration and wound healing. To understand the pathophysiology of ureteropelvic junction obstruction and determine why endopyelotomy fails in some obstructed ureteropelvic junctions, TGF-beta expression in obstructed and normal ureteropelvic junction segments was compared. MATERIALS AND METHODS: Immunohistochemical staining using a rabbit polyclonal anti-TGF-beta was performed on deparafinfized 4 microm. sections of paraffin blocked ureteropelvic junction segments. Human obstructed ureteropelvic junction segments were removed during primary pyeloplasties (11) and secondary pyeloplasties after endopyelotomy failure (11). Normal ureteropelvic junction segments were removed during nephrectomy for purposes unrelated to obstruction (11). Grading on a scale of 0 to 4 was performed by a physician blinded to the source of the specimen. RESULTS: Mean TGF-beta expression plus or minus standard error of the mean was significantly increased (p <0.02) in obstructed ureteropelvic junctions from primary pyeloplasties (2.6+/-0.7) compared to normal ureteropelvic junctions (1.6+/-0.7), as expected. However, TGF-beta expression in the endopyelotomy failure group (1.8+/-0.6) was not significantly different from that in normal ureteropelvic junctions and was significantly lower (p <0.05) than that in obstructed ureteropelvic junctions from primary pyeloplasties. CONCLUSIONS: Obstructed ureteropelvic junctions in cases of endopyelotomy failure have decreased expression of TGF-beta compared with other obstructed ureteropelvic junctions. These data suggest that an elevation of TGF-beta in obstructed ureteropelvic junctions may be necessary for successful tissue repair after endopyelotomy.
PURPOSE: The tumor suppressor gene WT-1 encodes a nuclear deoxyribonucleic acid binding protein that is a transcriptional regulator. This gene is commonly deleted or defective in Wilms tumors and the Denys-Drash syndrome. Recently WT-1 was demonstrated to be essential for the development of the urogenital tract. We determined whether we could induce WT-1 expression in mature kidneys induced to grow by performing contralateral nephrectomy in mature rats. MATERIALS AND METHODS: Northern analysis with a 32phosphorus-labeled antisense riboprobe synthesized by in vitro transcription of a 731 bp complementary deoxyribonucleic acid insert spanning exons 1 to 7 of the rat WT-1 in a pT7 Blue vector was used to demonstrate the expression of WT-1 in the developing and adult Sprague-Dawley rat kidney. RESULTS: Transcript levels of WT-1 in the rat kidney decreased from day 0 (day of birth) to day 16, after which WT-1 transcripts were undetectable in the normal rat kidney. Unilateral nephrectomy in the adult male Sprague-Dawley rat (250 to 300 gm.) induced the expression of WT-1 ribonucleic acid in the contralateral kidney to detectable levels by Northern analysis 0.25 hours after nephrectomy. Subsequently levels of WT-1 ribonucleic acid decreased progressively to undetectable by 3 hours after nephrectomy. Expression of this gene was not detected in the normal kidneys of adult rats or sham operated adult rats. CONCLUSIONS: These data suggest that the WT-1 gene product is involved in normal renal growth in the adult and developing rat kidney.
Functional delta opioid receptors were expressed in Xenopus oocytes following injection of poly(A+)RNA isolated from the mouse neuroblastoma X rat glioma hybrid cell line, NG108-15. Oocytes coinjected with in vitro transcribed beta 2-adrenergic receptor mRNA and NG108-15 cell mRNA had 3-fold elevated cAMP levels following isoproterenol treatment. Application of delta opioids to these oocytes caused dose-dependent inhibition of isoproterenol-induced increase of cAMP, which was antagonized by naltrexone. This is the first demonstration of the expression of opioid receptors in Xenopus oocytes. The ability to assay expression of delta opioid receptors functionally coupled to the inhibition of adenylate cyclase in Xenopus oocytes following injection of exogenous mRNA will provide a system to investigate the relationship of molecular structure to function of opioid receptors.
To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.
The studies described demonstrate that rat brain mRNA directs the synthesis of at least four types of functional EAA receptors in the Xenopus oocyte system, whereas in this system NCB-20 cell mRNA directs the synthesis of only the NMDA type of EAA receptor. The NMDA channel expressed in the oocyte, using either rat brain mRNA or NCB-20 cell mRNA, exhibits the pharmacologic properties of the neuronal receptor, including the functional association with the PCP receptor located within the NMDA-gated channel. The demonstration that mRNA isolated from NCB-20 cells lacking functional NMDA-activated channels, but bearing PCP binding sites, can encode functional NMDA-activated channels in the oocyte indicates some defect or regulating step in posttranslational processing or insertion of the receptors into the plasma membrane in the cell of origin. This is the only cell line known to (1) have PCP receptors that appear to be associated with NMDA receptors and (2) provide a homogeneous, self-replicating population of cells that can be manipulated genetically and by changing the extracellular environment. Consequently, the NCB-20 cell line will be useful for the study of the NMDA receptor and its expression.
Expression of the putative Wilms tumor gene (WT-1) was studied to investigate its role in renal growth. Compensatory renal growth was induced in 35-day-old Yorkshire-swine by unilateral nephrectomy. The contralateral kidney was removed 0.5 to 72 hours following the initial operation and analyzed for WT-1 gene expression by Northern analysis. Compensatory renal growth was detectable by wet weight measurements at 12 hours following contralateral nephrectomy. WT-1 gene expression was detectable in the contralateral kidney as early as 0.5 hours following unilateral nephrectomy. Expression of this gene was not detected in normal kidney from swine of this age group or from sham operated swine. In addition, we demonstrated the expression of this gene in newborn rat, pig and human kidneys, as well as human fetal kidney, indicating its role in nephrogenesis. These data suggest that expression of the WT-1 gene is involved in the regulatory mechanisms that control different types of renal growth.
Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the bead columns. Our data suggest that different methacrylate polymers induce different changes in adsorbed fibrinogen, which may interfere with its interaction with platelets, and that platelet retention in a methacrylate bead column involves interaction of the COOH-terminal end of the gamma-chain of adsorbed fibrinogen with platelet GpIIb/IIIa receptors.
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The mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptors. We report here that Xenopus oocytes injected with NCB-20 cell poly(A)+ RNA express N-methyl-D-aspartate (NMDA)-activated channels and that these channels include the PCP receptor site. In injected oocytes, NMDA application evoked a partially desensitizing inward current that was potentiated by glycine, blocked by the competitive antagonist D-2-amino-5-phosphonovaleric acid, blocked by Mg2+ and by Zn2+, and blocked in a use-dependent manner by the PCP receptor ligands PCP and MK-801. There was little or no response to kainate or quisqualate (agonists of the other excitatory amino acid receptors), to gamma-aminobutyric acid (an inhibitory transmitter), or to glycine (an inhibitory transmitter as well as an allosteric potentiator of NMDA channels). Thus, NMDA/PCP receptors expressed from NCB-20 cell mRNA exhibit properties similar to those of the neuronal receptors. The absence of expression of other excitatory amino acid receptors in this system makes it particularly useful for study of NMDA-evoked responses without interference from responses mediated by other receptors. Moreover, NCB-20 mRNA may be an appropriate starting material for cloning the cDNA(s) encoding the NMDA/PCP-receptor complex.
In the mammalian central nervous system, the excitatory amino acid transmitter L-glutamate activates three pharmacologically distinguishable receptors, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate receptors. The present paper addresses the issue of whether these three receptors operate independent channels or whether they share channels that may have several conductance substates. The Xenopus oocyte provides a system for expression of exogenous mRNAs that permits detailed study of receptor structure and function. In oocytes injected with rat brain mRNA, NMDA has a stoichiometry of channel activation different from that for kainate and quisqualate. NMDA activates its own channels as indicated by simple summation or near-summation of currents evoked by NMDA with those evoked by quisqualate or kainate. Deviations from summation are ascribable to lack of selectivity in which an agonist at one receptor acts as a weak antagonist at another receptor. A further indication of separate channels is that block of NMDA channels by Mg2+ or phencyclidine has no effect on kainate or quisqualate responses evoked during the block. Interactions of kainate and quisqualate are more complex, but they can be explained by lack of complete specificity of these agonists for their own receptors.
Recent evidence suggest that the N-methyl-D-aspartate (N-Me-D-Asp) channel is functionally and structurally associated with the phencyclidine (PCP) receptor, which mediates the psychotomimetic effects of PCP, sigma opioids, and dioxalanes. To investigate the relationship between N-Me-D-Asp and PCP receptors on a molecular level, we injected mRNA isolated from adult rat brain into Xenopus oocytes. In injected oocytes N-Me-D-Asp application (with glycine) evoked a partially desentizing inward current that was potentiated by glycine and blocked by D-(-)-amino-5-phosphonovaleric acid (D-APV), by Zn2+ and, in a voltage-dependent manner, by Mg2+. These results show that the distinguishing features of rat brain N-Me-D-Asp channels are reproduced in this translation system. In addition, kainic acid elicited a nondesensitizing inward current at short latency, and quisqualate elicited a delayed oscillatory inward current, presumably mediated by a second-messenger system. Responses to glutamate had both short-latency and delayed components. The PCP derivative N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) blocked the N-Me-D-Asp-evoked current, and its potency was comparable to its binding affinity in rat brain membranes. Onset of block required the presence of antagonist. Antagonism was stereoselective in that the active ligand dexoxadrol was a more effective blocker than its relatively inactive stereoisomer levoxadrol. adrol. Other PCP receptor ligands, (+)SKF-10,047 and MK-801, also blocked. Potencies of compounds active at N-Me-D-Asp and PCP receptors in oocytes were comparable to those obtained previously in electrophysiological and binding assays on neural tissues. These results indicate the coexpression of neuronal PCP and N-Me-D-Asp receptors in Xenopus oocytes.
Opioid, sigma, and phencyclidine (PCP) receptors were characterized in the mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20. Quantitative receptor assays under equilibrium binding conditions with highly specific radioligands demonstrated the presence of delta, but not mu or kappa, opioid receptors on NCB-20 cell membranes. NCB-20 cells were shown to possess two distinct sites specific for sigma opioids and PCP derivatives. One site was labeled by (+)-[3H]N-allylnormetazocine [(+)-[3H]SKF-10,047] (Kd = 69 nM; Bmax = 4100 fmol/mg of protein). The rank order of potency of drugs at this site was (+)-3-(3-hydroxy-phenyl)-N-(1-propyl)piperidine [(+)-3-PPP] greater than haloperidol greater than (+)-SKF-10,047 greater than (+/-)-ethylketocyclazocine greater than (+/-)-bremazocine greater than N-[1-(2-thienyl) cyclohexyl]piperidine (TCP) greater than dexoxadrol. This site is similar in its ligand selectivity to the haloperidol-sensitive sigma receptor of rat brain. The other site was labeled by the potent phencyclidine derivative [3H]TCP (Kd = 335 nM; Bmax = 9300 fmol/mg of protein). This density is equivalent to approximately 60,000 sites/cell. The rank order of potency of drugs at this site was TCP greater than (+)-3-PPP greater than PCP greater than dexoxadrol greater than haloperidol greater than cyclazocine greater than levoxadrol greater than (+)-SKF-10,047; mu and delta ligands were inactive. This site is similar to the rat brain PCP receptor. The NCB-20 cell line is the only cultured cell line that has been demonstrated to have PCP receptors.
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