ABSTRACT
INTRODUCTION: Transplant recipients have a higher incidence of cancer compared with the general population. This increased risk is related to the intensity and chronicity of immunosuppression that these patients receive. In this report, we present a case of a heart transplant woman with endometrial cancer who was diagnosed six months after transplantation. CASE REPORT: A 49-year-old woman who had undergone a heart transplant was referred to our department in May 2002 for final treatment. The diagnosis of endometrial cancer was established on the basis of the histopathology findings of the fractional curettage. Her heart transplant had occurred six months before, as a result of idiopathic restrictive cardiomyopathy. The patient received triple immunosuppression with cyclosporin, azathioprine and prednisone and she displayed no signs of acute graft rejection features. Laparoscopically assisted vaginal hysterectomy with adnexa was performed without any complications. Duration of surgery was 85 minutes. The patient was operated on under general anesthesia and intraabdominal pressure was automatically maintained at 10 mmHg with a carbon dioxide insufflator (AESCULAP, Germany). Immunosuppressive therapy was continued without modification. The postoperative course was uncomplicated in our patient. No significant changes in heart rate or blood pressure were observed. The patient was discharged from the hospital on the 11th postoperative day. Microscopic appearance revealed Stage I endometrial cancer. The patient is in good physical condition with normal heart performance and without disease recurrence. CONCLUSION: In our opinion LAVH was a justifiable form of surgical management in the treatment of a heart transplant recipient with an early-stage endometrial cancer.
Subject(s)
Adenocarcinoma/diagnosis , Endometrial Neoplasms/diagnosis , Heart Transplantation , Adenocarcinoma/pathology , Adenocarcinoma/surgery , Diagnosis, Differential , Endometrial Neoplasms/pathology , Endometrial Neoplasms/surgery , Fallopian Tubes/surgery , Female , Humans , Hysterectomy/methods , Laparoscopy/methods , Middle Aged , Ovariectomy/methods , Vagina/surgerySubject(s)
Hospitals, Special/history , Urology/history , History, 19th Century , History, 20th Century , Humans , PolandABSTRACT
OBJECTIVES: To assess a coefficient of the prostatic blood supply in patients with prostate cancer or benign prostatic hyperplasia (BPH), and thus improve the diagnostic and prognostic variables needed to evaluate patients with prostate cancer. PATIENTS AND METHODS: The vascular-stromal coefficient was defined as the ratio of the blood vessel surface area and the surface area of the prostate, using power Doppler functions and transrectal ultrasonography. The index was calculated for the plane of the prostatic surface from the apex to the widest points of the peripheral zone, and for the plane across the highest density of blood vessels in the transition zone. A micobubble contrast agent was used to intensify the acoustic signals from prostatic vessels. The images were reconstructed in three dimensions. In all, 61 patients with BPH and 32 with confirmed prostate cancer were examined, and compared with a control group of young men. RESULTS: In the horizontal plane crossing the apex of the prostate, the vascular-stromal coefficient was 0.0054 for the controls, 0.0072 for BPH and 0.0067 for cancer. At the level of the highest density of blood vessels the respective values were 0.0083, 0.038 and 0.029. The respective values for the first plane after using the contrast agent were 0.025, 0.092 and 0.0365, and in the second level 0.04, 0.15 and 0.14. CONCLUSION: The vascular-stromal coefficient measured at both levels was higher in patients with BPH than in those with cancer or than in the controls. The prostatic vascular system can be computer-reconstructed in three dimensions, allowing an evaluation of asymmetry and other vascular abnormalities. The contrast agent effectively increases the vascular acoustic signals from the prostate, enhancing by a factor of five the visualization of blood vessels when using the power Doppler option.
Subject(s)
Neovascularization, Pathologic/pathology , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/blood supply , Aged , Humans , Male , Middle Aged , Neovascularization, Pathologic/diagnostic imaging , Prostatic Hyperplasia/diagnostic imaging , Prostatic Neoplasms/diagnostic imaging , Ultrasonography, DopplerABSTRACT
OBJECTIVE: To assess whether organ-confined prostate cancer not detected on routine transrectal ultrasonography (TRUS) can be detected using three-dimensional (3-D) power Doppler methods. PATIENTS AND METHODS: A preliminary trial of the use of TRUS with 3-D power Doppler image reconstruction was conducted in 116 patients being evaluated for lower urinary tract symptoms. Using a microbubble enhancing agent, an adequate horizontal plane allowed the simultaneous visualization and assessment of the symmetry of echogenicity and vascularization of the peripheral area of both prostate lobes, which is not possible in standard TRUS. The peripheral zone vascular asymmetry in the horizontal plane was also assessed retrospectively in those patients with pT2a,b prostate carcinoma. RESULTS: Stage pT2a,b prostate carcinoma was confirmed in 26 patients; in this group, two independent investigators confirmed the vascular asymmetry between the left and right peripheral zone, in the horizontal plane, in 22 patients, giving a sensitivity of 85%. CONCLUSIONS: Vascular asymmetry allows guided biopsy in lesions that are invisible using standard TRUS. Microbubble agents are an effective method of increasing the acoustic signals from small vessels of the prostate.
Subject(s)
Prostatic Neoplasms/blood supply , Adult , Humans , Male , Middle Aged , Neovascularization, Pathologic , Prostatic Neoplasms/diagnostic imaging , Sensitivity and Specificity , Ultrasonography, Doppler/standardsABSTRACT
OBJECTIVE: To analyse retrospectively kidney and ureteric injuries (the former often associated with multiple-organ trauma) and thus optimize diagnostic and treatment methods. PATIENTS AND METHODS: The records and details of kidney and ureteric injures treated between 1995 and 1999 in 61 urological departments in Poland were analysed. RESULTS: In all, 887 kidney injuries were analysed; blunt trauma comprised 97%, with most injuries classified as renal contusion and minor parenchymal damage (687 cases). Intravenous urography was used in 80% of the patients and computed tomography in only 20%. In all, 234 patients (26%) underwent surgery; nephrectomy was the most common surgical treatment, in 170 patients (73% of those undergoing surgery). Complications occurred in 9% of patients after conservative treatment and in 5% after surgery. Of the 452 ureteric injuries, 340 (75%) were iatrogenic, 81 (18%) blunt injuries and 31 (7%) open injuries. Of the iatrogenic injuries 73% occurred during gynaecological procedures, 14% in general surgery and 14% in urological procedures. The most frequent diagnostic method was intravenous urography (244 cases), with retrograde pyelography (98) and ureteric catheterization in 125. The diagnosis was established immediately during surgery in 104 patients. The most frequent surgical treatment was uretero-neocystostomy (213, 47%), the others being a Boari flap (113, 25%), end-to-end anastomosis (92, 20%), reconstruction with an ileal loop (30, 7%) and autotransplantation (four, 1%). CONCLUSION: In Poland, patients with blunt kidney injuries often undergo surgery, with nephrectomy the most frequent procedure. Computed tomography with the intravenous administration of contrast medium should be considered the diagnostic method of choice for kidney injures. Catheterization of the ureters before surgery and an indigocarmine intravenous infusion (to stain the urine) when a ureteric injury is suspected may reduce the rate of iatrogenic injury and improve the rate of intraoperative diagnosis. We suggest catheterizing the ureters in any doubtful case to avoid injury, because prevention is better than treatment.
Subject(s)
Kidney/injuries , Ureter/injuries , Humans , Kidney/surgery , Multiple Trauma/epidemiology , Nephrectomy/statistics & numerical data , Poland/epidemiology , Postoperative Complications/epidemiology , Retrospective Studies , Tomography, X-Ray Computed/methods , Ureter/surgery , Ureteroscopy/methods , Ureterostomy/methods , Urinary Catheterization/methods , Urography/methodsABSTRACT
OBJECTIVE: To assess the occurrence and treatment of posterior urethral (most often caused by traffic accidents and comprising half of all cases of urinary tract traumas) and anterior urethral injury (usually iatrogenic during catheterization or cystoscopy, or caused by blunt trauma, e.g. straddle injury or penetrating trauma). PATIENTS AND METHODS: The records and details of patients with posterior and anterior urethral trauma were analysed from 61 urological departments in Poland between 1995 and 1999. RESULTS: During the 5-year period there were 268 cases of posterior and 255 of anterior urethral injury; for the former, most occurred during traffic accidents, and of the latter 206 were iatrogenic (during catheterization or cystoscopy), of which 48 were accompanied by perineal injury. The posterior injuries were isolated or involved many organs, mainly pelvic bones. Both types of injuries were diagnosed using ascending urethrography, voiding cysto-urethrography and urethroscopy/endoscopy. Treatment for posterior injuries included cystostomy alone or cystostomy with abdominal drainage or perineal drainage; some patients also had their urethra reconstructed. The treatment for anterior injuries was conservative in 193 patients and surgical in 62. CONCLUSION: The early detection and appropriate surgical treatment of posterior and anterior urethral injury is crucial for the recovery of urethral function, and avoids many complications.
Subject(s)
Urethra/injuries , Wounds, Nonpenetrating/surgery , Wounds, Penetrating/surgery , Accidental Falls/statistics & numerical data , Accidents, Traffic/statistics & numerical data , Cystoscopy/adverse effects , Cystostomy/methods , Drainage/methods , Humans , Incidence , Length of Stay , Poland/epidemiology , Suture Techniques , Urethra/surgery , Urinary Catheterization/adverse effects , Wounds, Nonpenetrating/etiology , Wounds, Penetrating/etiologyABSTRACT
OBJECTIVE: To assess incidence of urinary bladder injuries (frequently associated with pelvic trauma and often iatrogenic) in Poland. MATERIALS AND METHODS: The records and details of urinary bladder injuries treated between 1995 and 1999 were analysed for 61 urological departments in Poland. RESULTS: During the 5-year period 512 patients had urinary bladder injuries; in 210 (41%) the injury was caused by a road traffic accident, in eight (2%) by compression (crushing injury) within the limits of the pelvic bones, in 40 (8%) by a fall from a height, in three by a gunshot wound and in the remaining 251 (49%) the injury was iatrogenic. Among the 261 bladder injuries that were not iatrogenic, 41 (16%) were associated with pelvic bone trauma. In 36 patients there was simultaneous injury of the urinary bladder and posterior urethra, constituting 14% of such injuries and 7% of all trauma cases. The iatrogenic injuries were in 98 patients (39%) in urological departments, in 130 (52%) women in gynaecological departments and in 23 (9%) on surgical wards. The injury was open in 102 patients (20%) and closed in 372 (73%); there was bladder contusion in 38 patients (7%). The injuries were intraperitoneal in 225 patients (44%) and extraperitoneal in 287 (56%). For diagnosis, abdominal ultrasonography was used in 455 (89%) patients, intravenous pyelography in 266 (52%), cystography in 388 (76%) and computed tomography in 15 (3%). The delay between trauma and diagnosis was 0.5-124 h. Surgical treatment of the injury comprised a monolayer suture of the bladder wall in 51 patients (10%), a two-layered suture in 461 (90%), perivesical drainage in 468 (91%) and inspection of the peritoneal cavity in 232 (45%). The mean (range) interval between diagnosis and treatment was 14 (7-70) days. Seven patients died after the treatment failed. CONCLUSIONS: Almost half the patients had iatrogenic injuries, of which over half occurred in gynaecological and maternity wards. Thus it is important teach the basic range of urological operations to trainee doctors. The commonest diagnostic method was not ascending cystography but ultrasonography; we recommend ascending cystography be used with at least two views after filling the urinary bladder with approximately 300 mL of contrast medium, with an additional film after emptying the contrast medium. In patients with pelvic bone trauma it is reasonable to use spiral computed tomography with virtual analysis before surgery. A two-layered suture of the bladder wall with perivesical drainage should be used.
Subject(s)
Iatrogenic Disease/epidemiology , Urinary Bladder/injuries , Accidental Falls/statistics & numerical data , Accidents, Traffic/statistics & numerical data , Female , Health Surveys , Humans , Male , Poland/epidemiology , Urinary Bladder/surgery , Wounds, Gunshot/epidemiology , Wounds, Nonpenetrating/epidemiologyABSTRACT
OBJECTIVE: To present early experiences in Poland with high-energy transurethral microwave thermotherapy (HeTUMT), considered as a serious alternative to surgery in managing benign prostatic hypertrophy (BPH). PATIENTS AND METHODS: Sixty-one patients were treated in two centres using a commercial TUMT system. The patients were assessed at 5-6 weeks, 3 and 6 months after treatment, evaluating subjective complaints, the International Prostate Symptom Score (IPSS), urinary flow rate and postvoid residual volume. RESULTS: A complete follow-up was available in 44 patients; most had symptomatic improvement 3 months after HeTUMT but it was slightly less pronounced after 6 months. The improvement in objective variables increased up to 6 months after HeTUMT. There was > or = 50% improvement in subjective complaints in 43% of the patients, in urinary flow rate in 25% and in residual urine in half. Two patients required re-treatment for a urethral stricture and ineffective HeTUMT. CONCLUSIONS: HeTUMT may be a useful treatment for BPH but the outcome for an individual patient seems to be unpredictable. The indications for TUMT should be reviewed individually for each patient.
Subject(s)
Hyperthermia, Induced/methods , Microwaves/therapeutic use , Prostatic Hyperplasia/therapy , Transurethral Resection of Prostate/methods , Adult , Aged , Aged, 80 and over , Follow-Up Studies , Humans , Male , Middle Aged , PolandABSTRACT
Urological complications constitute significant problem following renal transplantation. Incidence ranges from 4 to 14% in graft recipients. The most important aspects concerning these complications are early diagnosis and prompt treatment, any delay in diagnosis and management may lead to deterioration of renal graft function or graft loss. The following case report discusses management of hydronephrosis in renal graft caused by ureter stenosis due to scarring and fibrosis of its distal end after remote kidney transplantation. The patient was a 33-year-old woman with previous history of end stage renal failure in the course of chronic glomerulonephritis. A triple drug immunosuppressive regimen consisting of Azathioprine (AZT), Cyclosporine A and Encorton (AZT + CsA + Encorton) was administered during a period of three years after kidney transplantation. At this time AZT administration was discontinued due to chronic viral hepatitis type B. Episodes of expansion sensation (discomfort) and graft pain were reported by the patient which after 3 days were followed by a period of oliguria and then anuria. The patient was admitted to the Department of Nephrology CMUJ, where ultrasound imaging revealed graft hydronephrosis. In the presence of such clinical and biochemical indications due to acute graft failure, one hemodialysis session, was performed. The patient was transferred to the Urological Department CMUJ where ureter exploration was attempted, but was unsuccessful. Subsequently percutaneous nephrostomy was performed which lead to immediate diuresis. Next, distal ureter stenosis (located by the urinary bladder) was surgically removed and reimplantation of the ureter was carried out. Due to early diagnosis and surgical reconstruction of the transplanted ureter, renal graft function returned to normal requiring only one hemo-dialysis session.
Subject(s)
Glomerulonephritis/complications , Hydronephrosis/surgery , Kidney Failure, Chronic/surgery , Kidney Transplantation/adverse effects , Nephrostomy, Percutaneous , Ureteral Obstruction/surgery , Adult , Chronic Disease , Female , Graft Rejection/therapy , Humans , Hydronephrosis/etiology , Kidney Failure, Chronic/etiology , Renal Dialysis , Ureteral Obstruction/etiologySubject(s)
Kidney/injuries , Wounds, Nonpenetrating/therapy , Wounds, Penetrating/therapy , Algorithms , Humans , Poland/epidemiology , Reoperation , San Francisco/epidemiology , Tomography, X-Ray Computed , Urography , Wounds, Gunshot/classification , Wounds, Gunshot/epidemiology , Wounds, Gunshot/surgery , Wounds, Nonpenetrating/classification , Wounds, Nonpenetrating/diagnosis , Wounds, Nonpenetrating/epidemiology , Wounds, Penetrating/classification , Wounds, Penetrating/diagnosis , Wounds, Penetrating/epidemiologyABSTRACT
Interleukin-2 (IL-2) is a powerful drug for treating cancer. However, it is only powerful if it is properly applied. That is, IL-2 should be applied at the tumor site, because at the transition of normal and malignant tissue are the tumor infiltrating cells. These should be activated by IL-2. Local application implies that IL-2 can be used in relatively low doses. It is becoming clear that even a single injection of IL-2 can cure cancer. IL-2 can also enhance the therapeutic effects of irradiation and Cisplatin. Locally applied IL-2 therapy is virtually non-toxic.
Subject(s)
Interleukin-2/administration & dosage , Neoplasms/therapy , Animals , Carcinoma, Squamous Cell/therapy , Cattle , Cisplatin/therapeutic use , Combined Modality Therapy , Dose-Response Relationship, Drug , Eye Neoplasms/therapy , Humans , Injections, Intralesional , Lymphoma/therapy , Mammary Neoplasms, Experimental/therapy , Mast-Cell Sarcoma/therapy , Mice , Mice, Inbred DBA , Neoplasm Transplantation , Time Factors , Tumor Cells, Cultured , Urinary Bladder Neoplasms/therapyABSTRACT
PURPOSE: We evaluate the therapeutic effect of intravesical interleukin-2 (IL-2) on T1 papillary bladder carcinoma after incomplete transurethral resection. MATERIALS AND METHODS: After incomplete transurethral resection we treated 10 patients in whom the marker lesion was left in place with 3 x 10(6) Chiron units IL-2 in 50 ml. saline plus 0.1% human serum albumin. The solution remained in the bladder for 2 hours and it was instilled on 5 consecutive days. The effect of IL-2 treatment on the marker lesion was evaluated by cystoscopy and repeat biopsy of the marker site 2 months after treatment. In addition, the effect on the recurrence of bladder tumors was studied. RESULTS: At 2 months 8 of the 10 marker lesions (80%) had completely regressed and there were no tumor cells on repeat biopsy. Four patients remained tumor-free after 30 to 54 months. We noted no toxic effects. In 1 patient with a 7-year history of bladder cancer the marker was only partially regressed after 2 months. After removal of the marker this patient remained tumor-free at a followup of 54 months. CONCLUSIONS: To our knowledge this report represents the first study of the effect of IL-2 on marker lesions left in place after transurethral resection. The results indicate that IL-2 instillations are feasible, and the combination of transurethral resection and IL-2 instillation may have a powerful antitumor effect. The therapeutic effects may not simply be due to intravesical IL-2, because previous transurethral resection probably caused some influx of infiltrating cells and the marker may have had tumor associated antigens. Consequently these effects may be due to the interaction of tumor associated antigens, infiltrating cells and IL-2.
Subject(s)
Carcinoma, Papillary/drug therapy , Interleukin-2/administration & dosage , Urinary Bladder Neoplasms/drug therapy , Administration, Intravesical , Adult , Aged , Carcinoma, Papillary/pathology , Female , Follow-Up Studies , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/epidemiology , Neoplasm Staging , Urinary Bladder Neoplasms/pathologyABSTRACT
Plasma membranes of rat platelets produced at normal platelet counts and during early recovery from immune-mediated thrombocytopenia were examined for the contents of carbohydrates, lipids and glycosphingolipids. Glucosylceramide, two monosialo-gangliosides and one disialo-ganglioside were found to be the major glycosphingolipids of platelets. During thrombocytopenia the contents of these glycosphingolipids as well as of ceramides were several fold elevated. Among carbohydrate constituents of platelets and platelet plasma membranes, glycogen content was increased and that of sialic acid decreased. These results are discussed in the light of literature data on relevant biochemical characteristics of megakaryocytes at different stages of maturation and on thrombopoiesis during acute experimental thrombocytopenia.
Subject(s)
Blood Platelets/metabolism , Ceramides/blood , Glycogen/blood , Glycosphingolipids/blood , N-Acetylneuraminic Acid/blood , Thrombocytopenia/blood , Animals , Cell Membrane/metabolism , Male , Rats , Rats, Wistar , Thrombocytopenia/immunologyABSTRACT
The discriminating and prognostic value of the expression of ABH(0) and TF antigens in tumour cells of 76 patients with urinary bladder tumours was evaluated. In patients with superficial tumours the most frequently defined phenotype was ABH(-) TF(+) and in those with deep tumours ABH(-) TF(-). The lowest frequency of recurrences was seen in patients with TFCr(+) antigenic phenotype and no patient died in this group. The mean lifespan of patients with TF(-) tumours was shorter (21 months) than of those with TF(+) tumours. For detection of tumours with higher risk of recurrences an analysis of the combination of markers revealed to be more predictive than analysis of a single marker.
Subject(s)
ABO Blood-Group System/immunology , Antigens, Neoplasm/biosynthesis , Antigens, Tumor-Associated, Carbohydrate/biosynthesis , Biomarkers, Tumor/biosynthesis , Isoantigens/biosynthesis , Urinary Bladder Neoplasms/immunology , Female , Humans , Male , Prognosis , Survival Analysis , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/mortalityABSTRACT
Beta human chorionic gonadotrophin levels have been assessed in blood serum of 79 patients with bladder tumours before and seven days after transurethral electroresection (TUR). With the growth grade of anaplasia and staging the mean serum beta HCG level increased. Beta HCG was a good biological marker to differentiate between superficial and deep tumours.
Subject(s)
Biomarkers, Tumor/blood , Chorionic Gonadotropin/blood , Peptide Fragments/blood , Urinary Bladder Neoplasms/blood , Aged , Chorionic Gonadotropin, beta Subunit, Human , Electrosurgery , Female , Follow-Up Studies , Humans , Male , Neoplasm Recurrence, Local/epidemiology , Neoplasm Staging , Postoperative Care , Preoperative Care , Prognosis , Survival Rate , Urinary Bladder Neoplasms/mortality , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/surgeryABSTRACT
Medical documentation of 80 women operated on because of ectopic pregnancy between the years 1991-1992 was analyzed. Methods of early diagnosis and ways of surgical approaches were discussed. Authors of this article give priority to controlling beta sub-unit HCG acc. to Romero, to ultrasonographic vaginal probe examination and to diagnostic laparoscopy. Early surgical intervention increases the chances to maintain woman's fertility. All procedures on women with ectopic--oviduct pregnancy should be sparing, depending on the ward equipment and personnel training and should be performed by the means of operative laparoscopy method.
Subject(s)
Pregnancy, Ectopic/diagnosis , Adult , Animals , Fallopian Tubes , Female , Humans , Laparoscopy , Pregnancy , Pregnancy, Ectopic/surgeryABSTRACT
Clinical-cytogenetic correlations were assessed in 79 patients with transitional cell carcinomas of the bladder to whom cytogenetic analysis of the primary tumor had been successful. High-grade (G2+G3) and high-stage (T2-T4) tumors had in general a higher ploidy level and contained more marker chromosomes, including multiple markers, compared to low-grade (G0+G1) and low stage (Ta+T1) tumors. The early recurrence rate (within 7 months) was significantly higher (p<0.05) in patients whose tumors contained marker chromosomes compared to cases without markers. Also, a significantly higher proportion of patients with marker chromosomes died due to their disease during the study period (follow-up time 30 months) compared to those without markers (84% versus 16%, p<0.005). The results confirm and extend previous studies showing that cytogenetic findings may be an important prognostic indicator in bladder cancer patients.
ABSTRACT
Five deletion mutants of the D/E linker region of the troponin C central helix were tested for conformational and functional differences from wild-type troponin C. The mutants were in the region 87KEDAKGKSEEE97: dEDA, dKG, dKGK, dKEDAKGK, and dSEEE, designed to change the length of the central helix and the orientation of the Ca(2+)-binding domains relative to each other [Dobrowolski, Z., Xu, G.-Q., & Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710]. Previous work showed that all mutants except dSEEE are partially defective in one part of the Ca2+ switch or the other. All mutants undergo Ca(2+)-dependent conformational changes as detected by changes in electrophoretic mobility, alpha-helix content, and hydrophobic exposure. Deletions of the central helix do not extensively alter the thermal stability of troponin C, as determined by temperature-dependent loss of alpha-helix. There are differences among the mutants that do not correlate with function. All troponin C mutants show Ca(2+)-dependent interaction with troponin I and T in polyacrylamide gels. Troponin I-troponin C interaction was also analyzed by Ca(2+)-dependent increase in the monomer/excimer ratio of tropinin I and relief of inhibition of the actomyosin S1 ATPase. While all mutants retain basic function, dKGK, dKEDAKGK, and dEDA have altered interaction with troponin I in the absence of Ca2+. dSEEE differs in conformation from wild type, but it is normal in functional assays. This conserved region of the D/E linker is not required for interaction with troponin I in the presence or absence of urea.
Subject(s)
Mutation , Troponin/genetics , Amino Acid Sequence , Calcium/metabolism , Circular Dichroism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Hot Temperature , Molecular Sequence Data , Myosins/antagonists & inhibitors , Protein Conformation , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Troponin/metabolism , Troponin C , Troponin IABSTRACT
Mutations have been made in the exposed region of the avian troponin C central helix, the D/E linker, which change its length and the orientation of the Ca2(+)-binding domains relative to each other. The region 87Glu-Asp-Ala-Lys-Gly-Lys-Ser-Glu-Glu-Glu97 has been altered in five deletion (d) mutants: dEDA, dKG, dKGK, dSEEE, and dKEDAKGK. The recombinant troponin Cs were expressed in Escherichia coli, purified, and assayed for function. All mutants retained basic troponin C function. They all bound Ca2+ to the low and high affinity sites, and they all were able to confer Ca2+ sensitivity on the regulated actomyosin ATPase. However, the regulatory function of all mutants except dSEEE was defective in one part of the Ca2+ switch or the other. In certain conditions dKGK and dKEDAKGK failed to inhibit fully whereas dEDA and dKG failed to activate the regulated actomyosin ATPase fully. The following general conclusions have been made. (a) The length of the D/E linker per se (assuming the linker is helical) and the orientation of the two Ca2(+)-binding domains relative to each other are not crucial for regulation. (b) The conserved charge cluster 95Glu-Glu-Glu97, in a region of troponin C known to bind to troponin I and postulated to be required for regulation, appears to be unimportant for function. (c) Deletion of 88Glu-Asp-Ala90 resulted in a troponin C that could not activate the actomyosin (or S1) ATPase over the level of actomyosin alone, thus defining a role for troponin C in this aspect of thin filament regulation. The results have been interpreted in terms of the crystallographic structure of troponin C and related to results with analogous calmodulin mutants.