Cloning of the mouse insulin receptor substrate-1 (IRS-1) gene and complete sequence of mouse IRS-1.

The mouse IRS-1 gene has been cloned and its structure determined. Mouse IRS-1 differs from rat by the absence of the potential C-terminal nucleotide binding site. Otherwise, the predicted IRS-1 protein is highly conserved between mouse, rat and humans, especially in the possible phosphorylation sites. The highly conserved nature of IRS-1 suggests the importance of these domains in the function of IRS-1 or its association with other proteins.

Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and regulation of the mouse insulin receptor substrate gene promoter.

To evaluate the potential for regulation of the insulin receptor substrate IRS-1, we have cloned the mouse IRS-1 gene, identified its promoter, and analyzed promoter activity in the basal state and in response to stimulation. The 5'-region of the mouse IRS-1 gene lacks typical CAAT and TATA boxes but contains nine potential Sp1 binding sites consistent with a housekeeping gene. The 5'-region of the IRS-1 gene also has significant regions of homology with the promoters of the progesterone receptor gene, the insulin-like growth factor I receptor gene, and the androgen receptor gene. Multiple transcription start sites were identified 0.4-1.2 kilobases (kb) upstream from the start codon. Using a chloramphenicol acetyl transferase assay in Chinese hamster ovary (CHO) cells, basal promoter activity was present in the 3.2 kb 5'-flanking region of IRS-1 gene. Within this region, there were 184-base pair and 60-base pair negative regulatory elements at -3.2 kb and -1.6 kb surrounded by positive elements. By gel shift assay, a nuclear factor was identified in CHO cells which binds to -1606 and -1586 sequence in the negative regulatory element and appears to be distinct from C/EBP, CREB, and AP-1. In 3T3-F442A adipocytes dexamethasone treatment significantly decreased IRS-1 mRNA and IRS-1 protein. This was due to a decrease in the half-life of IRS-1 mRNA, with no change in IRS-1 promoter-chloramphenicol acetyl transferase activity. Insulin also decreased IRS-1 protein by approximately 60% within 9 h but did so without altering IRS-1 mRNA levels or chloramphenicol acetyl transferase activity. Thus, both insulin and dexamethasone down-regulate IRS-1 expression at the posttranscriptional level; with insulin this is probably due to an effect on protein half-life, whereas with dexamethasone the effect is due to a change in the half-life of IRS-1 mRNA.

Efficacy and safety of acarbose in insulin-treated patients with type 2 diabetes.

OBJECTIVE: To demonstrate the efficacy, tolerability, and safety of acarbose compared with placebo in patients with type 2 diabetes inadequately controlled with diet and insulin. RESEARCH DESIGN AND METHODS: A multicenter randomized double-blind placebo-controlled parallel-group comparison study was conducted. The trial was 26 weeks with a 2-week screening period and a 24-week period of treatment with acarbose or placebo, with forced titration from 25 mg t.i.d. to 50 mg t.i.d. after 4 weeks, and titration of 50 mg t.i.d. to 100 mg t.i.d. after 12 weeks based on glucose control. The dosage of insulin was to remain stable. The primary efficacy variable was mean change from baseline in HbA1c, and secondary efficacy variables included mean changes in fasting and postprandial plasma glucose and triglyceride levels. RESULTS: The addition of acarbose to the treatment of patients receiving background insulin and diet therapy resulted in a statistically significant reduction in mean HbA1c of 0.69% compared with placebo. There were statistically significant reductions in postprandial plasma glucose and glucose area under the curve, and in postprandial serum triglyceride levels in the acarbose-treated patients. Gastrointestinal side effects were more frequently reported in the acarbose-treated patients. There were no significant differences in hypoglycemic events or liver transaminase elevations between groups. CONCLUSIONS: This study demonstrated that the addition of acarbose to patients with type 2 diabetes who are inadequately controlled with insulin and diet is safe and generally well tolerated and that it significantly lowers HbA1c and postprandial glucose levels.

Novel immunohistochemical markers of human renal allograft dysfunction--antithrombin III, Thy-1, urokinase, and alpha-smooth muscle actin.

We have studied the expression of alpha-smooth muscle actin (alpha sm-1) by mesangial cells, and the expression of Thy-1 glycoprotein, antithrombin III (ATIII), and urokinase by tubular epithelial cells in normal kidneys and dysfunctional renal allografts. Kidney biopsies were studied immunocytochemically for changes in each of these markers and the findings were classified into two groups and compared with creatinine plasma levels at the time the biopsies were taken. In dysfunctional grafts, mesangial alpha sm-1 and tubular epithelial Thy-1 reactivities were greatly diminished, and urokinase and ATIII were missing from proximal renal tubular epithelial cells. Urokinase, which was absent from normal renal glomeruli, appeared in glomeruli of some dysfunctional allografts. The possible usefulness of these markers in patient evaluations was supported by our finding that the distribution of vinculin, fibronectin, myosin, actin B4, desmin, glomerular HLA-DR, and the tubular expression of CD15 remained unchanged. These data prompt us to suggest that the immunocytochemical localization and evaluation of alpha sm-1, Thy-1, ATIII, and urokinase in kidney allografts may be useful adjuncts in the assessment of function in renal allografts.

The endothelial heparan sulfate-antithrombin III natural anticoagulant pathway in normal and transplanted human kidneys.

This is the first study of the antithrombin III-heparan sulfate natural anticoagulant pathway in human kidneys. Immunocytochemical experiments were done to demonstrate the pathway on normal renal endothelial cells. Enzymatic studies were done to show that the antithrombin III was anchored to endothelium by molecules of heparan sulfate. Displacement studies were done with glycosaminoglycans to show that the antithrombin III was bound to its glycosaminoglycan anchor via a heparinlike binding site, and replacement studies showed that antithrombin III could be returned to the same endothelial cells from which it was displaced. Immunocytochemical studies of biopsies showed that normally functioning renal allografts manifested the endothelial antithrombin III-heparan sulfate anticoagulant pathway. The pathway was compromised or absent from the microcirculation of biopsies from rejecting or rejected renal allografts, and the diminishment of endothelial ATIII was associated with the presence of fibrin deposition. It is concluded that compromise of the antithrombin III-heparan sulfate natural anticoagulant pathway results in compromised renal function in transplanted kidneys.

Tissue factor in normal and transplanted human kidneys.

Tissue factor (TF) plays a central role in the initiation of blood coagulation that frequently is enhanced in renal allografts. The identification and localization of TF was studied immunocytochemically in biopsies from normal and transplanted human kidneys and classified according to its distribution. The clinical status of each allograft was then correlated with the TF classifications. From these correlations, four distributional types of TF were identified. In normal kidneys, TF was localized to glomerular epithelium and basement membranes. Glomerular TF expression did not colocalize with mesangial or endothelial HLA-DR reactivity as determined by double antibody techniques. Tissue factor in donor kidneys also was identified in the renal capsule and in the adventitia of large arteries. These structures were not reactive in long-term transplanted grafts. Some cadaver kidneys prepared for transplantation had depleted glomerular TF, and exhibited TF reactivity within stromal tissues. Long-term allografts with progressive loss of renal function and kidneys with advanced rejection exhibited diminished TF reactivity of glomerular epithelium and basement membranes. This was frequently associated with fibrin deposition within the glomeruli and in the intertubular microcirculation. These findings indicate that the evaluation of TF in transplanted kidneys is related to the prognosis of graft survival.

Tubular antithrombin at transplantation determines subsequent renal allograft function.

BACKGROUND: Antithrombin is found in the microvasculature and tubules of normal and transplanted human kidneys. Although depletion of vascular antithrombin is associated with renal allograft dysfunction, neither the distribution nor clinical significance of tubular antithrombin is known. METHODS: Changes in tubular antithrombin in biopsy specimens (n=41) obtained from donor kidneys at transplantation were studied immunohistochemically. The relationship between these changes and subsequent graft function was analyzed. RESULTS: Granular intracellular antithrombin was found only within the proximal tubular epithelium. Allografts with depleted tubular antithrombin at transplantation (n=20) had significantly greater plasma creatinine concentrations at posttransplant days 3 (P < 0.001) and 5 (P < 0.03) than allografts with normal tubular antithrombin (n=21). Indeed, depletion of tubular antithrombin at transplantation correlated with the degree of graft dysfunction at 3 days after transplantation. CONCLUSIONS: Depleted tubular antithrombin at transplantation is associated with reduced early graft function, and this may identify patients at risk of a complicated follow-up.

OKT3 treatment of steroid-resistant renal allograft rejection.

The monoclonal antibody, Orthoclone OKT3 (OKT3), has been used with great efficacy in a prospective multicenter trial as therapy for first rejection episodes in cadaveric donor (CD) renal allograft recipients treated with azathioprine (AZA) and prednisone (P). However, although almost all rejection episodes were reversed, recurrent rejection occurred in approximately two-thirds of OKT3-treated patients in this earlier trial; infections also occurred in about two-thirds of patients, often related to the additional immunosuppression necessary to reverse the rerejection episodes. In the current series of patients, OKT3 was used to treat rejection in CD renal graft recipients in a protocol differing from the multicenter trial in two respects: baseline immunosuppression was cyclosporine (CsA) and P or CsA, AZA, and P (probably more potent immunosuppressive combinations than the AZA and P in the multicenter trial); and OKT3 treatment was reserved for rejection episodes resistant to 3 bolus infusions of methylprednisolone (MP), 5-10 mg/kg, rather than as primary therapy for first rejection episodes. Using this protocol, 46 of 74 rejection episodes (62%) diagnosed between 3/85 and 3/86 in CD renal allograft recipients were treated successfully with MP. Of the remaining 28 steroid-resistant rejection episodes, 27 (96%) were reversed with a 7-14-day course of OKT3, 5 mg/day. Only 5 recurrent rejection episodes (19%) have been observed in the 2-14-month follow-up period after OKT3 treatment; infections have occurred in 10 patients (36%), and three grafts (11%) have been lost in OKT3 treated patients. These results suggest that recurrent rejection and subsequent infection after OKT3 is used to treat rejection may be reduced in a protocol where CD renal allograft recipients are treated with baseline immunosuppression regimens including CsA and where OKT3 is reserved for steroid-resistant rejection. This approach appears to be both more cost-effective than, and as effective therapeutically as, treating all first rejection episodes with the monoclonal antibody.

Elective conversion from cyclosporine to azathioprine in recipients with stable renal function 6 months after kidney transplantation.

The average cost of cyclosporine over the first 6 months after renal transplantation has been $2450/recipient for recipients with stable renal function. Fifty-nine percent of all patients transplanted in 1984 do not have a third-party payment mechanism for outpatient medicines and many cannot afford cyclosporine. The expense of cyclosporine has, thus, mandated developing a protocol for conversion from cyclosporine to azathioprine. Using a protocol, which included a short overlap of cyclosporine and azathioprine and a temporary, modest increase in prednisone dose, 27 renal allograft recipients with stable renal function have undergone conversion of their immunosuppressive regimen approximately 6 months posttransplant with a minimum follow-up of 4 months from conversion. There has been no graft loss or patient death. Mean serum creatinine has been reduced in recipients with stable function after conversion (1.4 mg/dl 3 months postconversion compared to 1.8 mg/dl preconversion). However, acute breakthrough rejection has occurred in four recipients (15%), and, after reversal of rejection, mean serum creatinine is elevated (3.1 mg/dl) in this group. Only a single patient developed an infection during the conversion period. Thus, a policy of conversion from azathioprine appears to be a reasonable compromise for those patients who cannot afford long-term outpatient treatment with cyclosporine.

Mechanism of plasma catecholamine increases during coronary artery bypass and valve procedures.

Nineteen patients undergoing coronary revascularization (14 patients) or valve replacement (five patients) were studied to monitor the catecholamine levels following operation and for each of three consecutive postoperative days. A significant (p less than 0.01) elevation was observed in both the serum norepinephrine and epinephrine levels immediately following operation, with both responding in a similar fashion (r = 0.804, p = 0.016). While epinephrine returned to control levels within 3 days of operation, the norepinephrine levels remained above control. The elevation in catecholamines associated with cardiac operation is presumed to be secondary to (1) the stress of surgical trauma and (2) the influence of cardiopulmonary bypass, with its attendant hypothermia and hemodynamic alterations. A comparative analysis was performed of (1) the type of operation, (2) the sex of the patient, (3) the presence of postoperative arrhythmia, (4) the history of receiving propranolol, (5) the duration of cardiopulmonary bypass, (6) the duration of operation, and (7) the volume of fluid gradient absorbed during operation. None of these parameters except the sex of the patient was significantly related to the change in either norepinephrine or epinephrine associated with the operation. There was a significantly (p less than 0.05) higher epinephrine level at day 2 and 3 following operation in women than in men. None of the 19 patients had postoperative complications except for arrhythmias, which developed in nine patients and were not associated with the catecholamine responses. It is concluded that peak catecholamine stimulation does not reflect the ease of postoperative recovery. The duration of bypass and operation were also not directly related to the level of stimulation. It is apparent that there are multiple factors which combine to influence catecholamine secretion during cardiopulmonary bypass.

Perfusion technology in the hypothyroid patient.

Cardiopulmonary bypass may, by necessity, have to be performed in patients who are frankly hypothyroid. In treating five such patients, all of whom required coronary revascularization, it was noted that fluid balance during perfusion was considerably different than that in the normal population. In order to attempt to evaluate this difference, ten consecutive euthyroid patients having revascularization and the five hypothyroid patients were compared to correlate all fluid absorbed and excreted with the duration of bypass, the serum sodium, and subsequent weight gain. Fluid intake, urine output, and retained fluid were significantly elevated in the hypothyroid as compared to the euthyroid group, while serum sodium following operation was not significantly different. While there are considerable data indicating that hypothyroidism is associated with abnormal salt and water excretion, there is no information concerning the alterations which occur during cardiopulmonary bypass. The present study indicates that hypothyroidism is associated with significant diuresis (without administration of exogenous diuretic agents during cardiopulmonary bypass). The proposed explanation for this diuresis rests with the assumption that with cardiopulmonary bypass and appropriate fluid administration, the contracted blood volume in hypothyroid patients expands acutely and a diuresis results.

Osmotic separation of pancreatic exocrine cells from crude islet cell preparations.

A novel approach is introduced here to selectively lyse exocrine cells in an islet preparation by hypo-osmotic treatment. Time to hypotonic cell lysis required for the islet cells was much longer than that for the exocrine cells, which permits a possibility of selectively killing the exocrine cells by hypotonic treatment. The first set of experiments was designed to select an appropriate osmolality for the hypotonic treatment. Kinetic changes in cell volume in response to extracellular anisosmolalities (30 to 90 mOsm/kg) were recorded using an electronic particle counter. The results indicated that, when exposed to a 30 mOsm/kg solution, islet cells swelled slowly to reach volumetric equilibrium in approximately 3 min. There was no significant hypotonic cell lysis observed even at the end of 4 min (n = 4). In contrast, pancreatic exocrine cells, when exposed to the same solution, expanded rapidly to the lytic volume and burst within 30 s. Significant exocrine cell lysis was invariably achieved within 30 s when cells were exposed to the osmolalities below 60 mOsm/kg. For osmolalities between 70 to 80 mOsm/kg, exocrine cell lysis was highly variable. When cells were exposed to 80 to 90 mOsm/kg, no significant cell lysis was observed. Thus, an osmolality of 50 mOsm/kg is recommended for hypotonic treatment, as it maximizes the lysis of exocrine cells without unnecessarily stressing (osmotically) the islet cells. The second set of experiments (time-course experiments, 20 to 120 s) was designed to determine the length of exposure time for which the exocrine cells were irreversibly damaged but the islet cells had only swollen to such a degree that cell function is restored upon returning to an isotonic condition. Viability of the hypotonic treated cells was evaluated at two different levels: membrane integrity, measured by combined fluorescent dye staining with propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), and mitochondrial function, measured by colorimetric MTT assay. The results showed that hypotonic treatment in a 50 mOsm/kg solution for 30 s resulted in over 85% loss of the membrane integrity for the exocrine cells. About 90% of these membrane lysed cells lost mitochondrial function (n = 3). By contrast, under the same treatment, less than 15% of the islet cells lost membrane integrity and mitochondrial function (n = 3). In conclusion, hypotonic treatment with a 50 mOsm/kg solution for 20 to 30 s at room temperature is sufficient to lyse the majority of the contaminating exocrine cells in an islet cell preparation, while maintaining function in the islet cells.

Implications of a non-lamellar lipid phase for the tight junction stability. Part II: Reversible modulation of transepithelial resistance in high and low resistance MDCK-cells by basic amino acids, Ca2+, protamine and protons.

The transepithelial resistance of confluent epithelial cell monolayers was monitored to investigate the influence of basic amino acids, Ca2+, protamine and protons on tight junction electrical resistance. In an accompanying paper we investigated the effect of these substances on the lamellar/hexagonal II phase transition in reconstituted phospholipid membranes containing phosphatidylserine and phosphatidylethanolamine. We conclude that the permeability of tight junctions may be described by a lipid phase equilibrium where the lamellar phase corresponds to an open state and the hexagonal lipid phase to the closed state of the cell contact. This dynamic lipid model is well suited to describe the morphological as well as functional properties of the tight junctions.

Potent immunosuppression overcomes immunologic high-risk factors in recipients of cadaveric renal allografts.

With the introduction of more potent immunosuppressive regimens, increasing numbers of kidney transplant recipients traditionally viewed as being at high immunologic risk for rejection and graft loss have been accepted. These include recipients of multiple grafts, sensitized patients as measured by high panel reactive antibody (PRA), and patients with current warm B or historical positive crossmatches. Since November 1983, all recipients of cadaver kidneys have been treated with cyclosporine and prednisone. In addition, most also received a short posttransplant course of antilymphocyte globulin and long-term azathioprine. With these regimens, retransplantation, sensitization, current B-cell crossmatch and historical B- and/or T-cell crossmatch do not affect graft survival.

Stented Lich-Gregoir ureteroneocystostomy: case series report and cost-effectiveness analysis.

UNLABELLED: Extravesical ureteroneocystostomy to reestablish urinary tract continuity in renal transplantation has been examined through a meta-analysis of more than 14,000 kidney transplants leading to the finding that stented anastomosis was associated with a lower urologic complication rate compared with nonstented anastomoses. Fourteen stents must be used to prevent one urologic complication. We now report the urologic complication rate in our case series in which a stented Lich-Gregoir anastomosis was routinely utilized. We present a cost-effectiveness analysis regarding the usage of ureteral stents. METHODS: The records of 395 consecutive renal transplants were reviewed. Minimum follow-up time was 6 months. The standard anastomosis was a Lich-Gregoir with a 6- or 8-F 12- or 14-cm J-J stent. Monitored urologic complications included postoperative vesicoureteral leak or ureteral necrosis, obstruction or stricture, or clinically significant hematuria. Charges in 2004 US dollars were reported by the hospital accounting office. RESULTS: Four urologic complications were noted-three leaks and one stricture (complication rate of 1.0%). There were no stent-related complications requiring reoperation. There were no cases in which the urologic complication led to graft loss or patient death. Total charges associated with stent use were $1,087 per patient, or $15,218 per urologic complication prevented. CONCLUSIONS: The urologic complication rate in this case series is similar to the five previously published randomized trials, as well as our previously published meta-analysis. These results support the routine use of a ureteral stent. Our analysis suggests that stent use is cost effective.

Maintenance of adequate hemodialysis access. Prevention of neointimal hyperplasia.

The maintenance of adequate hemodialysis vascular access is frequently complicated in the patient with polytetrafluoroethylene (PTFE) A-V hemodialysis grafts by venous anastomotic stenosis. This stenosis is caused by neointimal hyperplasia (NIH), a response to vascular injury. In this study, the authors prospectively analyzed the effect of a short-term regimen consisting of administration of two medications, heparin and low molecular weight dextran, on the development of NIH at the venous anastomosis in 79 patients with PTFE A-V hemodialysis grafts. In addition, they evaluated other parameters' effects on the development of NIH. In comparison with control subjects, heparin had some effect in minimizing the development of NIH in the PTFE grafts when evaluated radiologically at 3 months, although this effect was not statistically significant. Low molecular weight dextran, however, had no trend or statistically significant effect on this venous anastomotic narrowing. Interestingly, patient age, use of calcium channel blockers, and presence of diabetes mellitus (DM) all appeared to affect the development of NIH. Increasing age and use of calcium channel blockers was associated with decreased development of NIH; conversely, DM was associated with worsened NIH. In evaluation of access survival (time to first access failure), degree of venous anastomosis stenosis at 3 months was not predictive. Patient time on dialysis pre graft placement was the only measured parameter related to access failure. The method of dialysis pre graft placement (hemodialysis versus peritoneal dialysis) was not a significant factor in early access failure. Pharmacologic treatment of venous anastomotic narrowing in PTFE hemodialysis grafts due to NIH continues to be difficult. Short-term treatment with the tested medication failed to statistically affect NIH. Patient age, use of calcium channel blockers, and presence of DM were all factors in the development of NIH. Of measured parameters, time on dialysis pre graft placement was the only factor correlated with early access failure. In future treatment regimens, one should consider more prolonged treatment. In addition, noted risk factors should be considered when determining type of renal replacement therapy.

Spiritual nursing interventions.

Oncology, parish, and hospice nurses in the Midwest were surveyed to explore what nursing interventions they implement to enhance the spirituality of clients and how they learned about these interventions. Some 95 spiritual nursing interventions were identified and ranked according to frequency of implementation. The most frequently identified nursing interventions were referral, prayer, active listening, facilitation and validation of clients' feelings and thoughts, conveying acceptance, and instilling hope. The most frequently ranked implemented interventions were communication and religious nursing interventions. Only 15% of the participants indicated that they learned the most about spirituality and spiritual nursing interventions from basic and advanced nursing education combined. Findings support the need for increased emphasis of theoretical and clinical spiritual knowledge in nursing education and the development of a typology of spiritual nursing interventions to direct nurses in enhancing the spirituality of clients and implementing holistic nursing care.

[Ewing's sarcoma of the ethmoid bone. Case report]. / Sarcome d'Ewing de l'ethmoïde. A propos d'un cas.

Ewing's sarcoma is rarely located at the base of the skull. We report a case of ethmoid Ewing's sarcoma. Diagnosis was established on microscopic examination. A complete surgical resection was performed, followed by cranial base reconstruction. During the postoperative period, a combined course of chemotherapy and radiotherapy was performed. We also present a review of the literature.

[Ewing's sarcoma of the head and neck: a case report]. / Sarcome d'Ewing et localisations ORL: à propos d'un cas.

We report a case of Ewing's sarcoma located in the ethmoid sinus. The patient complained of facial pain, anosmia and visual defect. Diagnosis was established at microscopic examination with histoimmunochemistry and molecular biology. Treatment combined chemotherapy and surgical resection with skull base reconstruction and post-operative ratio and chemotherapy.