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PubMed1998

Intratubular application of sodium azide inhibits loop of Henle reabsorption and tubuloglomerular feedback response in anesthetized rats.

Sodium azide (NaN3, AZ) is a potent inhibitor and uncoupler of oxidative phosphorylation as well as a nitrovasodilator after being converted to nitric oxide (NO). We studied the effect of intratubular application of AZ on loop of Henle reabsorption and tubuloglomerular feedback (TGF) employing renal micropuncture experiments in nephrons with superficial glomeruli of anesthetized Munich-Wistar-Fromter rats. During perfusion of Henle's loop downstream from an obstructing wax block, AZ (3x10(-5) mol/l and 3x10(-4) mol/l) concentration-dependently increased early distal tubular flow rate and sodium and potassium ion concentration (V(ED), [Na+]ED, [K+]ED). In comparison, application of furosemide (10(-4) mol/l), the action of which is restricted to the water-impermeable thick ascending limb of Henle's loop (TALH) and the macula densa, similarly increased [Na+]ED and [K+]ED, but did not affect V(ED). The effect of AZ on loop of Henle reabsorption appeared to be predominantly localized upstream to the TALH since (1) AZ significantly inhibited net fluid reabsorption (the latter being completely abolished at 3x10(-4) mol/l), (2) the effect of AZ on [Na+]ED and [K+]ED could be mimicked by perfusing the Henle's loop at a flow rate that caused a comparable increase in V(ED) (reflecting a comparable load to TALH), and (3) the effects of AZ and furosemide were additive. In spite of the increase in [Na+]ED and [K+]ED, intratubular application of AZ caused a concentration-dependent inhibition of TGF response, the latter being assessed as the fall in early proximal tubular stop flow pressure during perfusion of Henle's loop at increasing flow rate. Like AZ and furosemide, the NO donor sodium nitroprusside (10(-4) mol/l) blunted the TGF response, but in contrast to furosemide or AZ, it caused a minor decrease in V(ED), without changing [Na+]ED or [K+]ED. The inhibitory effect of AZ on TGF was abolished by the NO scavenger carboxy PTIO. In summary, AZ inhibits both reabsorption in the water-permeable segment of Henle's loop and the TGF response. The effect on reabsorption may be linked to metabolic inhibition rather than NO release, whereas the blunted TGF response appears to involve conversion to NO.

Absorption↗
PubMed1990

Tight junction regulation during impaired ion transport in blind loops of rat jejunum.

Epithelial cell tight junction structure in self-filling blind loops of rat jejunum, a model for blind loop syndrome in humans, was analyzed morphometrically along the crypt-villus axis. In control jejunum, the number of strands and junctional depth, including meshwork depth, decreased from crypt to villus tip. In the blind loop, aberrant strands appeared below the meshwork, particularly in crypt cells. Consequently, total junctional depth was greater than in controls. Furthermore, strand number and junctional meshwork depth were increased in blind loops at the villus tip. It is that site along the crypt-villus axis which showed the most shallow junction in control jejunum. This structural change is paralleled by a three-fold increase in epithelial resistance as previously measured by alternating current impedance analysis. Relative Na over Cl permeability (PNa:Cl) was obtained from dilution potential measurements. PNa:Cl was 1.50:1 in control jejunum and 1.35:1 in the blind loop (n.s.). Considering the cation selectivity of the tight junction, the increase in epithelial resistance in blind loops cannot be attributed to a collapse of the lateral intercellular space but is due to changes in tight junctional permeability resulting from structural alteration. The blind loop syndrome represents a further example of diminished epithelial ion transport and concomitant decrease in tight junction permeability, thus supporting the general concept of regulation of the tight junction in response to active transport activity.

Animals↗
PubMed1984

Technetium-99m HIDA hepatobiliary scanning in evaluation of afferent loop syndrome.

A study of 118 patients, operated on with Billroth II gastrectomy for peptic disease and affected by postgastrectomy syndromes, was carried out. Fifty patients were investigated by means of technetium-99m HIDA hepatobiliary scanning. In 18 patients, in whom an afferent loop syndrome was clinically suspected, hepatobiliary scanning demonstrated an altered afferent loop emptying in 8 and atonic distension of the gallbladder without afferent loop motility changes in 10. Among the patients in the first group, four were treated with a biliary diversion surgical procedure and in the second group, two patients underwent cholecystectomy. Our findings indicate that biliary vomiting, right upper abdominal pain pyrosis, and biliary diarrhea in Billroth II gastrectomized patients are not always pathognomonic symptoms of afferent loop syndrome. Technetium-99m HIDA hepatobiliary scanning represents the only diagnostic means of afferent loop syndrome definition. A differential diagnosis of abnormal afferent loop emptying and gallbladder dyskinesia is necessary for the management planning of these patients, and furthermore, when a surgical treatment is required, biliary diversion with Roux-Y anastomosis or Braun's biliary diversion seems the treatment of choice for afferent loop syndrome, whereas cholecystectomy represents the best procedure for atonic distension of the gallbladder.

Adult↗
PubMed2025

Spatial Mapping and Interactome Profiling of m6A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling.

m6A-Modified R-loops (m6A-R-loops) play crucial roles in epigenetic regulation and genome stability, yet resolving their spatial distribution and protein interactomes in live cells remains challenging. To address this, we developed m6A-R-loop proximity labeling (m6A-RLPL), a chemically inducible split-APEX2 proximity labeling technology integrating dual-target recognition using the RNA-DNA hybrid binding domain of RNase H1 for R-loop targeting and m6A reader protein's YTH domain for m6A recognition, coupled with an abscisic acid (ABA)-inducible dimerization system for signal amplification. This technology revealed host m6A-R-loops enriched with nucleoli under normal conditions. When applied to herpes simplex virus (HSV) infection, it further demonstrated viral m6A-R-loops undergoing dramatic accumulation within phase-separated granules in replication compartments during late-stage infection. Proximity proteomics identified ZC3H4 and CCDC124 as essential regulators maintaining these structures, which serve as transcription sites for HSV late genes, with disruption selectively impairing viral transcription. m6A-RLPL establishes a generalizable approach for spatially resolved profiling of m6A-R-loop interactomes and organizational dynamics in living systems.

Humans↗
PubMed1979

Regional membrane specialization in the thin limbs of Henle's loops as seen by freeze-fracture electron microscopy.

Rat thin limbs of Henle were studied by freeze-fracture electron microscopy. Thin limb segments in both short- and long-looped nephrons were identified by previously developed ultrastructural criteria, continuity with known thick segments, and architectural relationships in the outer medulla. Intramembrane particle (IMP) density and the number of intramembrane fibrils comprising the zonula occludens were determined for each morphologically identifiable thin limb segment. The IMP density on the protoplasmic faces of both the luminal and abluminal membranes of the upper portion of the descending thin limb (DTL) of the long-looped nephron is quantitatively greater than in the short-looped thin limb, lower portion of the long-looped DTL, and in the ascending thin limb. The zonulae occludens in the long-looped upper DTL consists of a single fibril; the long-looped lower DTL contains 3.13 +/- 0.14 fibrils; the ascending thin limb contains 1.31 +/- 0.09 fibrils; and the short-looped DTL contains 3.75 +/- 0.19 fibrils. These studies further support the contention that there is anatomic heterogeneity among the thin limb segments. Because direct physiologic studies in the thin limbs are incomplete and conflicting, the need for correlative physiologic studies on anatomically characterized structures is indicated.

Animals↗
PubMed1975

Physical factors influencing fluid reabsorption from Henle's loop.

Our objective was to produce reductions in the luminal volume of Henle's loop and increases in linear flow velocity through the loop. We did this in a recollection micropuncture study by collecting fluid with and without suction from early distal tubules. With suction, transit time of fast green dye through the loop decreased by 34%, calculated loop volume decreased by 28%, and fractional water reabsorption fell from 73.6 to 70.3% (p smaller than 0.025) in water diuretic rats. Absolute water reabsorption did not decrease significantly. In urea-saline dieuretic rats transit time decreased 25%, calculated loop volume decreased 22%, fractional reabsorption fell from 59.0 to 51.7% (smaller than 0.001), and absolute reabsorption decreased by 2.3 nl/min (p smaller than 0.025). Single nephron glomerular filtration rate, distal tubular sodium concentration, and osmolality were unaffected. The less pronounced effect of collection with suction in water diuretic rats may be related to the lower medullary fluid osmolality, which was 338 plus or minus 9 (S.E.) mOsmol/kg as compared to 497 plus or minus 35 in urea saline diuretic rats. Collecting fluid with suction from late proximal tubules did not alter glomerular filtration rate or fractional water reabsorption. Stumpe et al. ((1970) J. Clin. Invest. 49, 1200-1212) noted an inverse correlation between fluid reabsorption from Henle's loop and flow velocity in rats with hypertension or congestive heart failure. One can reproduce this correlation by artificially altering the transmural pressure gradient in the loop.

Animals↗
PubMed1992

Increased loop chloride uptake precedes hypertension in Dahl salt-sensitive rats.

When studied at equivalent renal perfusion pressures, loop segment chloride reabsorption is greater in hypertensive Dahl salt-sensitive (S) than Dahl salt-resistant (R) rats. To determine whether this difference in loop reabsorption is present before the onset of hypertension, volume expanded and euvolemic Dahl rats maintained on low NaCl diets were examined using micropuncture and in vivo microperfusion techniques. Neuroendocrine differences between groups were eliminated by renal denervation and fixing plasma aldosterone, norepinephrine, and vasopressin levels. After volume expansion, urinary NaCl excretion was less in S than R rats. Early distal tubule fluid-to-plasma chloride and inulin (TF/PCl/IN) ratio and chloride delivery were also less while loop chloride reabsorption was greater in S rats. Proximal delivery was not different between groups. In euvolemia, urinary NaCl excretion and loop chloride reabsorption were not different between S and R rats. However, when loop chloride delivery was increased by microperfusion techniques, lower distal TF/PCl/IN ratios and greater loop chloride reabsorption were clearly demonstrated in euvolemic S rats. Thus loop chloride reabsorption is greater in S than R rats before the development of hypertension. This difference depends on increasing delivery rates for its manifestation.

Animals↗
PubMed1985

Renin secretion and loop of Henle chloride reabsorption in the adrenalectomized rat.

Renin release is increased in the adrenalectomized rat and is not inhibited by sodium chloride administration. The purpose of this study was to determine whether increased renin release is related to impaired absorptive chloride transport in the loop of Henle. Chloride transport in the loop was measured before and after acute saline infusion in three groups of rats: 1) saline-drinking adrenalectomized rats (Adx); 2) saline-drinking dexamethasone-treated adrenalectomized rats (Dex); and 3) water-drinking sham-operated controls. Unrelated to differences of arterial pressure, glomerular filtration rate, or net sodium chloride balance, chloride reabsorption in the loop of Henle of Adx [836 +/- 172 peq/min (SE)] was less (P less than 0.01) than in controls (1,646 +/- 353) and Dex (1,377 +/- 318) before saline infusion. After saline infusion, chloride delivery to the loop increased (P less than 0.05) in all three groups. However, loop chloride reabsorption increased (P less than 0.01) only in controls and Dex but not in Adx. Before saline infusion, plasma renin concentration (PRC) of Adx (350 +/- 108 U/ml) was greater (P less than 0.01) than that in controls (56 +/- 6) or Dex (108 +/- 36); sodium chloride infusion failed to inhibit PRC in Adx, whereas PRC was suppressed (P less than 0.01) by saline in Dex and controls. Thus stimulation of renin release in adrenalectomized animals was associated with decreased absorptive chloride transport in the loop of Henle. Dexamethasone normalized loop function and renin responsiveness to sodium chloride.

Absorption↗
PubMed1990

Urea transport in a distributed loop model of the urine-concentrating mechanism.

Continuously distributed loops of Henle were used in a central core model of the rat kidney's urine-concentrating mechanism to investigate the importance of overlapping loops for three different modes of urea transport in the long loops of Henle: 1) urea-impermeable loops, 2) urea-permeable loops (as indicated by perfused tubule experiments), and 3) loops with urea-permeable descending limbs and active urea transport out of thin ascending limbs. Mode 1 produces high papillary tip osmolality in accordance with tissue slice experiments, but the relative contribution of urea to the osmolality of the central core and the long descending limbs is below experimental measurements. Mode 2 generates no significant osmolality increase in the inner medulla, in agreement with other model studies. Mode 3 produces high papillary tip osmolality with a substantial contribution of urea to the osmolality of the core and the descending limbs, which is more in accordance with experiments. The results suggest that 1) overlapping loops may produce a cascade effect that contributes to the inner medullary concentrating mechanism and that 2) new experiments are needed to more certainly ascertain the urea transport characteristics of the thin ascending limbs.

Animals↗
PubMed1996

Role of 20-HETE in elevating loop chloride reabsorption in Dahl SS/Jr rats.

In vivo tubular perfusion experiments were performed in normotensive Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats maintained from birth on a low salt (0.4% NaCl) diet to examine the role of 20-HETE in elevating loop Cl- transport in SS/Jr rats. Chloride reabsorption in the loop of Henle was significantly greater in SS/Jr than in SR/Jr rats (77 +/- 2% versus 57 +/- 3% of the perfused Cl- load). When the renal metabolism of arachidonic acid by P450 was blocked by the addition of 17-octadecynoic acid (10 micromol/L) to the perfusate, loop Cl- transport increased in SR/Jr rats to 70 +/- 2% of the delivered Cl- load, but it had no effect in SS/Jr rats. Conversely, addition of 20-HETE (10 micromol/L) to the perfusate lowered loop Cl- transport in S rats to 60 +/- 2% of perfused Cl- load, but it had no effect in SR/Jr rats. Addition of another endogenously formed HETE to the perfusate, 15-HETE (20 micromol/L), had no effect on Cl- reabsorption in the loop of Henle of SS/Jr rats. These findings indicate that endogenously produced P450 metabolites of arachidonic acid regulate Cl- transport in the loop of Henle of the rat in vivo and support the view that a diminished production of 20-HETE in the outer medulla of SS/Jr rats contributes to the elevation in loop Cl- transport and the resetting of the pressure-natriuresis relation in these animals.

Animals↗
PubMed1995

The role of continuous infusion loop diuretics.

OBJECTIVE: To evaluate the role of continuous infusion loop diuretics in selected patient populations, discuss the advantages and disadvantages associated with continuous infusion, and recommend monitoring parameters for the use of continuous infusion therapy. Current dosing guidelines for continuous infusion loop diuretics have not been established, but a summary of previously studied doses is provided. DATA SOURCES: A literature search using MEDLINE, International Pharmaceutical Abstracts, as well as additional references found in pertinent articles. STUDY SELECTION AND DATA EXTRACTION: Clinical studies concerning the use of loop diuretics administered by continuous infusion were evaluated in selected patient populations. All articles and clinical studies were considered for possible inclusion in the review. Information judged to be pertinent by the authors was selected for discussion. DATA SYNTHESIS: Comparative studies in the congestive heart failure (CHF), renal-insufficient, and postcardiac surgery patient populations have shown that loop diuretics administered by continuous infusion are more beneficial than those given by intermittent bolus administration. In adult patients with CHF, furosemide 3-4 mg/h is recommended. In adult and pediatric postcardiac surgery patients, furosemide dosages of 0.05 and 0.1 mg/kg/h have produced diuresis. In patients with renal insufficiency, bumetanide 0.912 mg/h has produced diuresis. Intravenous bolus doses were used in all studies reviewed except 1. These studies have indicated that continuous infusion of the loop diuretics yields diuresis without increasing toxicity. CONCLUSIONS: The use of continuous infusion loop diuretics is a therapeutic alternative for patients requiring diuresis. This form of administration has provided more consistent urine flow, fewer alterations in fluid balances, fewer urinary losses of electrolytes as well as decreased dosage of the diuretic requirements. The disadvantages have not been fully elucidated because of the limited evaluation of this administration method. Few studies have used this method of administration; however, the few data available indicate that continuous infusion of loop diuretics is an efficacious alternative to conventional therapy.

Diuretics↗
PubMed2002

CT diagnosis of afferent loop syndrome.

OBJECTIVE: To report the computed tomography (CT) features of afferent loop syndrome which is often clinically unsuspected. MATERIALS AND METHODS: The CT studies of five patients with afferent loop syndrome were reviewed. These patients had undergone gastroenterostomy and other surgical procedures, for malignant neoplasms in four and for peptic ulcer in one. Patients presented between 4 months and 15 years (average 5.5 years) after surgery. Symptoms were acute in all patients, the most common symptom being abdominal pain. In addition three of the patients had chronic symptoms including recurrent episodes of abdominal pain, recurrent ascending cholangitis, jaundice and bilious vomiting. RESULTS: The obstructed afferent loop appeared on CT as a fluid-filled tubular mass with an average diameter of 5.3cm. Valvulae conniventes were identified in all, and small intraluminal air bubbles in four. The dilated afferent loop was opacified with oral contrast material in only one patient. The loop was located in the subhepatic area in three patients and crossed the midline between the aorta and the superior mesenteric vessels in the other two. Additional findings included biliary dilatation in all five patients and signs of pancreatitis in one. Treatment was surgical in four patients (delayed for four months in one) and conservative in one. CONCLUSION: A fluid-filled tubular structure containing small air bubbles in the right upper quadrant or crossing the midline on CT in symptomatic patients after gastroenterostomy is characteristic of a dilated, possibly obstructed, afferent loop. The diagnosis is often not suspected clinically since patients may present many years after the initial surgery. Recognition of the characteristic CT findings will avoid both inappropriate procedures such as aspiration or drainage of an obstructed afferent loop and delay in treatment.

Abdominal Pain↗
PubMed1991

Attenuation of azosemide's action in the loop of Henle of rat kidney by nonsteroidal anti-inflammatory drugs.

The purpose of the present study, which was performed in anaesthetized rats, was to clarify whether the nonsteroidal anti-inflammatory drugs (NSAIDs) indomethacin and meclofenamate interact with the diuretic action of the new loop diuretic azosemide (Luret, CAS-27589-33-9). Since azosemide's diuretic action shows a lower onset and a more prolonged duration as compared to classical loop diuretics it was also tested whether azosemide's tubular action is mediated by an active metabolite. The results demonstrate that azosemide, when infused directly into the lumen of superficial loops of Henle, dose-dependently diminished the loops sodium and chloride reabsorption. A half-maximum effect was observed at an azosemide concentration of 3 x 10(-6) mol/l. These results clearly prove that the diuretic effect of azosemide is exerted by the drug itself and does not require metabolism to an active metabolite. Luminal application of the drug also dose-dependently increased early distal sodium and chloride concentrations, indicating that the drug's tubular action is located in the thick ascending limb of Henle's loop. Indomethacin and meclofenamate blunted the diuretic, natriuretic and chloruretic response to azosemide, and microperfusion experiments on single loops of Henle revealed an attenuation by NSAIDs of azosemide's inhibitory action on the loops sodium and chloride reabsorption. The quantitative extent of this interaction was nearly identical to that observed previously for the NSAID-furosemide interaction.

Animals↗
PubMed1992

Studies of DNA dumbbells. III. Theoretical analysis of optical melting curves of dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

Optical melting curves of seven DNA dumbbells with the 16 base-pair duplex sequence 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' linked on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops measured in 30, 70, and 120 mM Na+ are analyzed in terms of the numerically exact statistical thermodynamic model of DNA melting. The construction and characterization of these molecules were described in the previous paper (Amaratunga et al., 1992). As was recently reported for hairpins (T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight, 1990, Biopolymers, Vol. 29, pp. 1715-1734) theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting the parameters representing loop and circle formation to optimize the fits. The systematically determined empirical parameters provide evaluations of the free energies of hairpin loop formation delta Gloop (n) and single-strand circles delta Gcircle (N), as a function of end loop size, n = 2-14, and circle size, N = 32 + 2n. The dependence of these quantities on solvent ionic strength over the range from 30 to 120 mM Na+ was evaluated. An approximately analytical expression for the partition function Q(T) of the dumbbells was formulated that allowed a means for determining the transition enthalpy delta H degrees and entropy delta S degrees for every dumbbell, revealing the dependence of these quantities on loop size. In this multistate approach a manifold of partially melted intermediate microstates are considered and therefore no assumptions regarding the nature of the melting transitions (that they are two-state) are required. The transition thermodynamic parameters were also determined from a van't Hoff analysis of the melting curves. Comparisons between the results of the multistate analysis and the two-state van't Hoff analysis revealed significant differences for the dumbbells with larger end loops, indicating that the melting transitions of the larger looped dumbbells deviate considerably from two-state behavior. Results are then compared with published melting studies of much larger DNA dumbbells (D. B. Naritsin and Y. L. Lyubchenko, 1990, Journal of Biomolecular Structure and Dynamics, Vol. 8, pp. 1-13), of small hairpins (Paner et al., 1990; M. J. Doktycz, T. M. Paner, M. Amaratunga and A. S. Benight, 1990, Biopolymers, Vol. 30, pp. 829-845) and another dumbbell (A. S. Benight, J. M. Schurr, P. F. Flynn, B. R. Reid, and D. E. Wemmer, 1988) Journal of Molecular Biology, Vol. 200, pp. 377-399).(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed2004

Oesophageal and gastric bile exposure after gastroduodenal surgery with Henley's interposition or a Roux-en-Y loop.

BACKGROUND: The degree which the various reconstruction techniques prevent bile reflux after gastroduodenal surgery has been poorly studied. METHODS: Bile exposure in the intestinal tract just proximal to the jejunal loop was measured with the Bilitec 2000 device for 24 h after gastroduodenal surgery in three groups of patients. Group 1 comprised 24 patients with a 60-cm Henley's loop after total gastrectomy. Group 2 included 31 patients with a 60-cm Roux-en-Y loop after total (22 patients) or subtotal (nine) gastrectomy. Group 3 contained 21 patients with a 60-cm Roux-en-Y loop anastomosed to the proximal duodenum as part of a duodenal switch operation for pathological transpyloric duodenogastric reflux. Bile exposure, measured as the percentage time with bile absorbance greater than 0.25, was classified as nil, within the range of a control population of healthy subjects, or pathological (above the 95th percentile for the control population). Reflux symptoms were scored and all patients had upper gastrointestinal endoscopy. RESULTS: Bile was detected in the intestine proximal to the loop in none of 24 patients in group 1, eight of 31 in group 2 and 12 of 21 in group 3 (P < 0.001). The mean reflux symptom score increased with the degree of bile exposure, and the proportion of patients with oesophagitis or gastritis correlated well with the extent of bile exposure (P < 0.001). CONCLUSION: A long Henley's loop was more effective in preventing bile reflux than a long Roux-en-Y loop. Bilitec data correlated well with the severity of reflux symptoms and the presence of mucosal lesions.

Adolescent↗
PubMed1993

Properties of bacteriorhodopsin derivatives constructed by insertion of an exogenous epitope into extra-membrane loops.

Bacteriorhodopsin (BR) is folded into a bundle of seven alpha-helices which is embedded in the cellular membrane of Halobacterium salinarium; these helices are connected by short extra-membrane loops, three on the cytoplasmic side and three on the outside. Oligonucleotide-directed insertion or replacement mutagenesis was used to integrate the C-terminal sequence (13 amino acids long) of Sendai virus L-protein individually into each of the six helix-connecting loops. The altered gene products were obtained by expression of the mutant genes in either Escherichia coli or Schizosaccharomyces pombe and were used to reconstitute BR in proteoliposomes. In four cases (altered loops B/C, C/D, D/E or E/F), the mutant BRs were found to be fully functional as judged by light-driven proton pumping and photocycle kinetics. Within the four functional BR variants, recognition of the viral epitope by a monoclonal antibody is restricted to modified loops B/C and E/F. Immunogold staining of S.pombe cells producing either of the two latter BR variants shows that the protein is distributed among various cellular membranes but is not present in mitochondrial membranes. Sequence alteration of loop A/B or F/G resulted in loss of function, most plausibly due to a folding defect of the respective proteins. These results on the one hand document differences in structural importance of the various BR extra-membrane loops and on the other hand open the door to the construction of multifunctional membrane proteins via loop replacement mutagenesis of BR.

Amino Acid Sequence↗
PubMed2001

Relation between the flexibility of the WPD loop and the activity of the catalytic domain of protein tyrosine phosphatase SHP-1.

The conserved WPD loop of protein tyrosine phosphatases play an important role in the catalytic activity and the invariant aspartate residue acts as a general acid/base catalyst in the dephosphorylation reaction. In our previous report, we have demonstrated that the catalytic activities of the PTPs are influenced by the flexibility and stability of the WPD loop in its active "open" conformation [Yang et al., 1998]. Phosphatases with a more flexible WPD loop generally have higher specific activity. In this report, we modify the WPD loop of SHP-1 by alanine-scan mutation of the residues flanking the loop and measure their effects on the catalytic activity of the phosphatase. We show that the S418A, V424A, S426A, E427A, and P428A mutants increase the phosphatase activity, possibly due to the increased flexibility of the WPD loop, whereas the L417A, L417G and P425A mutants decrease its phosphatase activity. In addition, we propose that the two-proline residues in the WPD loop (Pro(420) and Pro(425) in SHP-1) work as pivotal points through a conserved hydrophobic network and allows residues between the pivotal points to have maximum flexibility in enhancing the phosphatase activity. Furthermore, our data suggest that the hydrolysis of the phosphoryl-cysteine intermediate, not its formation, is the rate-limiting step with p-nitrophenyl phosphate as the substrate while both the steps are rate-limiting with phosphotyrosine as the substrate.

Alanine↗
PubMed1995

Regulation of autocrine gastrin expression by the TGF alpha autocrine loop.

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.

Animals↗