A multi-institutional analysis of laparoscopic orchidopexy.

OBJECTIVE: To combine and analyse the results from centres with a large experience of laparoscopy for the impalpable testis with small series, to determine the expected success rate for laparoscopic orchidopexy. METHODS: A questionnaire was distributed to participating paediatric urologists; each contributor retrospectively reviewed the clinical charts for their cases of therapeutic laparoscopy for an impalpable testis, detailing 36 variables for each patient. The data were collated centrally into a computerized database. For inclusion, the testis was intra-abdominal (including 'peeping' at the internal ring) at laparoscopic examination, was not managed through an open approach and did not undergo orchidectomy. Three surgical groups were assessed, with success defined as lack of atrophy and intrascrotal position: group 1, primary laparoscopic orchidopexy; group 2, a one-stage Fowler-Stephens (F-S) orchidopexy; and group 3, a two-stage F-S orchidopexy. RESULTS: Data were gathered from 10 centres in the USA, covering the period 1990-1999; 252 patients representing 310 testes were included and overall, 15.2% were lost to follow-up. There was no significant difference between success rates in the larger and smaller series. Atrophy occurred in 2.2% of 178 testes, 22.2% of 27 testes and 10.3% of 58 testes in groups 1-3, respectively. Testes were not in a satisfactory scrotal position in 0.6%, 7.4% and 1.7% of groups 1-3, respectively. The mean follow-up for each group was 7.7, 8.6 and 20.0 months, respectively. The overall success for all groups was 92.8% (97.2% group 1; 74.1% group 2; 87.9% group 3), with an atrophy rate of 6.1%. CONCLUSION: Laparoscopic orchidopexy for the intra-abdominal testis, in both large and small series, can be expected to have a success rate higher than that historically ascribed to open orchidopexy. Within this series, single-stage F-S laparoscopic orchidopexy resulted in a significantly higher atrophy rate than the two-stage repair. However, when considering both F-S approaches, the laparoscopic approach gave greater success than previously reported for the same open approaches. Despite the weaknesses inherent in a retrospective unrandomized study, we conclude that laparoscopic orchidopexy is, if not the procedure of choice, an acceptable and successful approach to the impalpable undescended testicle.

Laparoscopic augmentation cystoplasty using the novel biomaterial Surgisis: small-intestinal submucosa.

BACKGROUND AND PURPOSE: Urinary bladder augmentation is indicated for diverse conditions, including neurogenic bladder, cancer resection, spinal cord injury, and congenital anomalies. The ideal cystoplasty material is yet to be described. Native gastrointestinal segments commonly used are limited by leakage and small-bowel obstruction, metabolic/nutritional abnormalities, calculi, and malignancy. This study assessed laparoscopic bladder augmentation with porcine small intestinal submucosa (SIS). MATERIALS AND METHODS: Five female pigs (<25 kg) were prepared for surgery under general anesthesia. After Veress needle insufflation, a main 10-mm trocar was placed in the midline for the laparoscope, with two lateral 10-mm ports added for operative instruments. The bladder dome was incised, and a patch of SIS was sewn into the bladder using running 2-0 Vicryl. Three animals served as technical studies. Two additional sows underwent long-term survival surgery: one undiverted and one diverted via a Stamey suprapubic catheter. RESULTS: There were no operative losses. The mean operative time was 140 minutes. The SIS graft held the sutures without tearing. Laparoscopic survey revealed no urine leaks at bladder closure. All five animals voided postoperatively. Urinary extravasation was evident in the three undiverted technique animals. In the other two sows, cystoscopy at 7 days showed intact suture lines without evidence of urinary extravasation and with normal vesicular volumes. Tissue growth was evident, but the graft margins were still discernible. CONCLUSIONS: Laparoscopic bladder augmentation was possible using SIS but at minimal volumes. There were no operative complications; however, the material was difficult to deploy and may benefit from application of an absorbable scaffold. Postoperative urinary drainage is necessary. Further studies will optimize the graft configuration for maximal augmentation.

Characterization of ureteral dysfunction in an experimental model of congenital bladder outlet obstruction.

PURPOSE: Ureteral dysfunction is a significant sequela of congenital bladder outlet obstruction. However, the structural and functional alterations associated with ureteral dysfunction are not well defined. A model of fetal bladder obstruction in sheep was used to characterize the changes in ureteral smooth muscle, extracellular matrix (ECM) and functional properties in response to bladder outlet obstruction. MATERIALS AND METHODS: Partial bladder outlet obstruction was created in fetal sheep at gestational age 95 days via placement of a metal ring around the proximal urethra as well as ligation of the urachus. Ureters were harvested at 109 and 135 days (full term = 140 days) to determine the relative composition of smooth muscle, ECM and urothelium by morphometric analysis and to measure DNA and protein concentrations. Ureteral tissue from 135 day gestation obstructed and control sheep was harvested and immediately placed in Krebs solution. Smooth muscle strips (2-3 mm. x 7-8 mm.) were suspended in organ baths. The frequency and amplitude of spontaneous ureteral contractions was as well as the response to electric field stimulation (EFS) were determined. RESULTS: Bladder outlet obstruction caused a significant increase in ureteral weight, smooth muscle mass and total ECM at both 109 and 135 days gestation. Total ureteral DNA was greater in obstructed compared with sham ureters at 135 days gestation. Obstructed ureters demonstrated greater amplitude and frequency of spontaneous contractions as well as more pronounced response to EFS when compared to sham ureters. CONCLUSIONS: The fetal ureter responds to bladder obstruction with smooth muscle hyperplasia and hypertrophy which is associated with increased spontaneous activity and augmented response to EFS. ECM content is markedly increased indicating a shift in the balance of connective tissue synthesis and degradation. Congenital post-obstructive ureteral dysfunction therefore appears to be the result of dysregulated smooth muscle cell growth and altered ECM homeostasis producing abnormal ureteral contractility.

Renal renin-angiotensin system dysregulation caused by partial bladder outlet obstruction in fetal sheep.

BACKGROUND: To determine whether fetal renal obstruction activates the renal renin-angiotensin system (RAS), an important mediator in normal kidney development and obstructive nephropathy, we used a model of fetal partial bladder outlet obstruction (PBOO). METHODS: Total RNA and protein was extracted from kidney of sheep fetuses with partial bladder outlet obstruction created at 95 days gestation, after 2 (N = 6) and 5 weeks of obstruction (term; N = 6), and from normal fetal sheep at various time points between 60 and 135 days of gestation (total N = 19). Relative levels of mRNA for renin, angiotensinogen, type 1 and 2 angiotensin II (Ang II) receptors (AT-1 and AT-2), and transforming growth factor-beta1 (TGF-beta1) were assessed by semiquantitative reverse transcription-polymerase chain reaction. Expression levels of AT-2 receptor protein were measured by Western blot analysis. RESULTS: Renin mRNA expression was increased (250%) after two weeks of obstruction. In normal fetuses, AT-1 expression was low at 60 to 75 days of gestation and increased toward the end of gestation, whereas AT-2 expression showed a reversed pattern. At 109 days, PBOO caused an increased expression of AT-2 mRNA compared with normals (400%). Correspondingly, AT-2 receptor protein was more abundant in obstructed kidneys. TGF-beta1 mRNA expression was significantly increased in obstructed kidneys at 109 days gestation. CONCLUSIONS: These observations confirm the reciprocal developmental regulation of AT-1 and AT-2 receptors' expression, suggesting their functional role in renal development. Partial bladder outlet obstruction produces specific alterations: increased renin expression and altered balance of receptor subtypes, which may induce altered functional and vascular regulation of the obstructed fetal kidney. TGF-beta1, a mediator of Ang II-induced fibrosis, may play a role in inducing and propagating interstitial fibrosis.

Pediatric retroperitoneoscopic nephrectomy using 2 mm. instrumentation.

PURPOSE: We describe several modifications of the retroperitoneoscopic approach to nephrectomy for benign renal disease, including the use of 2 mm. instrumentation and prone patient positioning. MATERIALS AND METHODS: A total of 14 children underwent retroperitoneoscopic nephrectomy in the prone position. An inflatable dissecting device was inserted into the retroperitoneum after a small muscle splitting incision was made at the lateral border of the sacrospinalis muscle approximately 1 cm. below the costovertebral angle. After inflation the dissecting device was replaced with a 5 mm. cannula and pneumoretroperitoneum was maintained with carbon dioxide insufflation. Two 2 mm. trocars were then placed under endoscopic guidance. Dissection was performed using 2 mm. instrumentation and the specimen was retrieved through the largest port site. RESULTS: Nephrectomy was performed in 9 girls and 5 boys 3 months to 9.8 years old. The preoperative diagnosis included chronic pyelonephritis with minimal renal function, reflux with a nonfunctioning kidney, multicystic dysplastic kidney, an upper pole dysplastic moiety with an associated ureterocele and a dysplastic kidney with a vaginal ectopic ureter. Mean operative time for retroperitoneoscopic nephrectomy was 142 minutes with an estimated blood loss of less than 15 ml. Contralateral ureteral reimplantation was performed after retroperitoneoscopic dissection in 5 patients. Overall average hospital stay was 2 days and there were no complications. CONCLUSIONS: Several modifications of the retroperitoneal approach, including the use of prone patient positioning and 2 mm. instrumentation for visualization and dissection, may improve the safety and efficacy of this technique in children.

Fetal partial urethral obstruction causes renal fibrosis and is associated with proteolytic imbalance.

PURPOSE: We determine whether fetal bladder outlet obstruction induces renal fibrosis, and is associated with an alteration in the regulation of connective tissue degradation and the presence of fibrogenic interstitial cells. MATERIALS AND METHODS: Partial bladder outlet obstruction was surgically induced in 33 fetal sheep at 95 days of gestation. These animals and 24 normal age matched controls were sacrificed at 109, 116 and 135 (term) days of gestation, and the kidneys were rapidly retrieved, drained and weighed. Representative whole kidney samples were snap frozen for assessment of deoxyribonucleic acid, protein and collagen content. Morphometric analysis and alpha-smooth muscle actin immunohistochemistry were performed on histological specimens from formalin fixed kidneys. Tissue extract from fresh kidney specimens were analyzed for metalloproteinase and tissue inhibitor of metalloproteinase activity. Urine samples obtained at the time of sacrifice were analyzed for electrolyte, creatinine and N-acetyl glucosaminidase excretion. RESULTS: All obstructed kidneys were hydronephrotic and larger than age matched controls. Obstructed kidneys at term showed interstitial fibrosis, as measured by increased extracellular matrix volume fraction (45% in male obstructed kidneys versus 2.5% in normal male kidneys, p = 0.0004), increased total collagen content (120 mg./kidney in male obstructed versus 20 mg. in normal male animals, p = 0.016) and collagen/deoxyribonucleic acid content per kidney (2.78 versus 0.53 mg./mg., p = 0.016). Metalloproteinase-1 activity was significantly lower in obstructed kidneys (210 versus 380 U./mg. protein in normal kidneys). Tissue inhibitor of metalloproteinase activity was undetectable in both groups. The presence of an increased population of myofibroblasts often associated with fibrotic processes was seen by alpha-smooth muscle actin staining which was localized to interstitial cells throughout the cortex in obstructed kidneys. CONCLUSIONS: Fetal partial bladder outlet obstruction induces renal interstitial fibrosis as early as 2 weeks after obstruction. A possible mechanism for this process is a shift in proteolytic activity to reduce matrix degradation in obstructed kidneys. These changes might be mediated by the increased number of fibrogenic interstitial cells. The observations suggest several potential approaches to developing an understanding of congenital obstructive uropathy.

Experimental fetal vesicoureteral reflux induces renal tubular and glomerular damage, and is associated with persistent bladder instability.

PURPOSE: We assessed renal function and urodynamic status in animals with experimental congenital vesicoureteral reflux. MATERIALS AND METHODS: Vesicoureteral reflux was surgically induced in male sheep fetuses at 95 days of gestation. After birth the animals were maintained on antibiotic prophylaxis. At ages 1 week and 6 months reflux was assessed by fluoroscopic voiding cystography. Cystometrography was performed with the animals awake. Serum creatinine, inulin clearance and the excretion of urinary N-acetyl-beta-D-glucosaminidase were measured at ages 1 week, 1 month and 6 months by surveillance urine cultures. Urinary concentrating capacity was assessed by desmopressin testing at ages 1 and 6 months. RESULTS: Nine animals (18 renal units) were born after the induction of reflux. There was no reflux in 2 renal units, while reflux was mild in 2, moderate in 5 and severe in 9. In the 6 animals available for followup at age 6 months only severe reflux persisted. Reflux resolution was associated with normalization of bladder urodynamics. Surveillance urine cultures were negative until age 6 months, when infection developed in 3 of the 6 lambs. In all animals serum creatinine was normal during followup. Glomerular filtration rate in the lambs with reflux was no different from normal at age 1 week but it was significantly less than normal independent of infection at age 6 months (2.7 versus 3.9 ml./kg. per minute, p = 0.002). As an indicator of renal tubular injury the ratio of N-acetyl-beta-D-glucosaminidase-to-creatinine remained significantly higher in animals with reflux than in normal animals from ages 1 week to 6 months (51.0 versus 10.2 IU/mg., p = 0.03). Maximal concentrating ability after desmopressin testing was already less than normal by age 1 month with a maximal increase of 98 versus 435 mOsm./l. in lambs with reflux versus normal lambs (p <0.0001). It was further impaired by age 6 months. Urodynamic evaluation of the animals with reflux revealed decreased bladder compliance at age 1 week with normal voiding pressure. In addition, in those with reflux there was a more pronounced immature voiding pattern with multiple phasic contractions due to sphincteric activity as well as a post-void bladder contraction. CONCLUSIONS: Our model of fetal vesicoureteral reflux induces alterations in renal function that are consistent with clinical observations and marked by altered tubular function but a relatively mild decrease in glomerular filtration. Bladder dynamics are altered, consistent with observations in human neonates with high grade reflux and bladder instability. Whether this represents cause or effect remains unclear. Our model permits focused study of the interaction of these factors in neonatal reflux and may allow the application of more specific therapies, particularly those directed toward mechanisms of renal and bladder dysfunction.

Experimental vesicoureteral reflux in the fetus depends on bladder function and causes renal fibrosis.

PURPOSE: Prenatal diagnosis allows the early detection of vesicoureteral reflux in an increasing number of newborns, some of whom are born with impaired kidney function. This situation challenges our current understanding of the pathophysiology, natural history and, therefore, treatment of reflux. We created a fetal sheep model of vesicoureteral reflux to study the mechanisms of fetal reflux nephropathy. MATERIALS AND METHODS: Vesicoureteral reflux was induced in fetal sheep at 95 days of gestation (term 140 days) by open bladder incision of the intravesical ureteral tunnel. All animals underwent urachal ligation and in female subjects mild bladder outlet obstruction was created with a gold ring. RESULTS: At term reflux was detected in 18 of 28 renal units by filling cystography. Refluxing kidneys were hydronephrotic and larger than normal. At term mean kidney weight was 21.1 gm. (range 12.2 to 35.0) in male subjects with reflux compared to 8.5 gm. (range 6.5 to 11.3) in normal male subjects (p <0.001) and 11.5 gm. (range 8.5 to 15.8) in male subjects with urachal ligation only (p = 0.035). In female subjects there was no change in renal weight. Renal histology revealed a thin, structurally normal cortex with small subcortical cysts and a hypoplastic medulla with mesenchymal tissue replacing normal ducts. Total mean renal collagen content was significantly increased to 51.7 mg. (range 35 to 81) in the refluxing kidneys of male animals, while it was 23.8 mg. (range 12.1 to 38.4) in normal male animals (p = 0.03). The fractional excretion of sodium was elevated in refluxing kidneys based on sodium-to-creatinine ratios in bladder urine. CONCLUSIONS: In a novel model of fetal vesicoureteral reflux we showed that prenatal reflux nephropathy is characterized by altered renal growth regulation, structural maldevelopment without overt dysplasia, excess matrix deposition and impaired excretory function.

Current findings in diagnostic laparoscopic evaluation of the nonpalpable testis.

PURPOSE: We delineate the current findings and contribution of diagnostic laparoscopic evaluation in the management of nonpalpable testis. MATERIALS AND METHODS: We reviewed all cases in which laparoscopy was considered the management associated with a nonpalpable testis in a 4-year period. Since our previous series, we have performed a careful examination for the testis after induction of anesthesia but before committing to laparoscopy. We recorded testis position and quality, character of the vas deferens and spermatic vessels, type of management and contribution of laparoscopy. We also reviewed contemporary published series and collated the findings of studies performed elsewhere. RESULTS: We identified 263 nonpalpable testes in 225 patients between September 1992 and 1996. In 40 patients 46 testes (18%) were found during physical examination under anesthesia. Of the remaining cases considered appropriate for laparoscopy 215 with complete records were further analyzed. Only 12.6% could be considered missed on examination due to a viable testis distal to the inguinal ring. Of the testes 45.7% would have been found during inguinal exploration alone. In 9.8% of the patients there were intra-abdominal vanishing testes, while 4.2% had indeterminate cord structures on inguinal exploration that would have prompted abdominal extension without a laparoscopic demonstration that the vas and vessels entered the canal. A conventional inguinal incision would have provided optimal exposure for operative management in 34% of the testes. For testes distal to the internal ring when the vas and vessels were distinctly atretic we never identified a viable testis, while a normal appearing vas and vessel were associated with a 45% chance of a salvageable testis. Laparoscopy was informative regarding testis position in all cases in which it was performed. CONCLUSIONS: In 13.2% of the cases laparoscopic findings precluded unnecessary abdominal exploration. The typical surgical incision for inguinal exploration would have left the surgeon compromised in 66% of the cases compared to the approach optimized as a result of laparoscopic testicular localization. Of the patients 34% arguably did not benefit from laparoscopy versus inguinal exploration. A simple examination under anesthesia significantly decreases the number of uninformative laparoscopic evaluations, and it is well worth the cost of a few minutes of operative time.

Pneumoperitoneum produces reversible renal dysfunction in animals with normal and chronically reduced renal function.

Laparoscopic surgery with intraperitoneal insufflation is associated with acute oliguria. Although in healthy patients, this impairment is transient and without any apparent sequelae, as the scope of laparoscopic surgery expands, subtle renal injury may become clinically significant, particularly when applications expand to patients with reduced baseline renal function. We have investigated the changes in renal function during and after pneumoperitoneum in animals with reduced renal mass to identify both acute alterations and long-term impairments, if any. Twelve swine underwent surgical reduction in renal mass to produce chronic renal insufficiency. Glomerular filtration rate (GFR) was determined by inulin clearance for each animal before and after ablation to establish the degree of renal impairment (mean 22%; range 18%-31% of normal). The animals were followed during a stepwise insufflation as a study of pneumoperitoneum-induced changes in chronic renal failure. Urine output declined dramatically (-80% at 20 mm Hg), the GFR fell (-63% at 20 mm Hg), and renal blood flow declined (mean -12%; range -9% to -19%) over the course of the test. These values did not return to baseline during a 90-minute observation period after release of the pneumoperitoneum. Acute renal failure occurred despite aggressive hydration with maintenance of central venous pressure and only modest changes in cardiac output. The animals were exposed to a 6-hour CO2 pneumoperitoneum to 20 mm Hg to model the insult of complex laparoscopy. This exposure resulted in elevation of the amount of N-methyl-beta-D-glucosaminidase being shed into the urine in addition to the previously indicated impairments. The animals were allowed to recover for 1 week, and then GFR was again measured. The GFR returned to the preexposure chronic renal failure levels for both the group as a whole and individual animals. The magnitude and duration of the alteration in urine output, GFR, and renal blood flow suggest that regulatory mechanisms rather than simple mechanical forces are involved in the acute changes. No long-term impact on renal function from the acute renal injury was identified, even in animals with existing renal insufficiency.

Mechanism and role of growth arrest in programmed (apoptotic) death of prostatic cancer cells induced by thapsigargin.

BACKGROUND: More than 95% of metastatic androgen independent prostatic cancer cells per day are in a proliferatively quiescent G0 state [Berges et al.: Clin Cancer Res 1:473-480, 1995] limiting their responsiveness to anti-proliferative chemotherapeutic agents. Novel therapeutics capable of activating the programmed (apoptotic) death pathway in these cells without requiring entrance into the proliferative cell cycle are urgently needed. Thapsigargin (TG) treatment of rapidly growing androgen independent prostatic cancer cells arrests such cells in G0 and induces their programmed death. This raises not only the issue of the mechanism for such growth arrest, but also whether this programmed death is simply a response of rapidly growing cells to growth arrest making cytotoxicity still dependent upon the initial rate of cell proliferation. METHODS: To resolve the mechanism of TG induced growth arrest, rat AT3.1 prostatic cancer cells were analyzed for RNA and protein expression of the growth arrest gene, gadd153, intracellular free Ca2+ levels (Cai), and cell cycle distribution on exposure to TG alone and in combination with Ca2+ chelation induced by BAPTA-AM or BAPTA-AM/EGTA. To resolve whether growth arrest is required for TG cytotoxicity, primary cultures of proliferatively quiescent, human prostatic cancer cells were exposed to TG. RESULTS: Co-treatment of androgen independent AT-3 rat prostatic cancer cells with the Cai chelator BAPTA plus TG prevented growth arrest, as monitored by DNA flow cytometry, and failure to induce mRNA and protein for gadd153, demonstrating that growth arrest is due to Cai elevation, not depletion of intracellular Ca2+ pools. In addition, proliferatively quiescent G0 primary cultures of human prostatic cancer cells were resistant to anti-proliferative agents, but could be induced to undergo programmed death by TG as documented by morphological criteria and 14C-labeled DNA fragmentation assays. CONCLUSIONS: These results demonstrate that TG with its ability to elevate Cai induces proliferating prostate cancer cells to growth arrest. Such Cai dependent growth arrest is not required, however, since TG can induce the programmed death of proliferatively quiescent G0 prostatic cancer cells without requiring either growth arrest or progression through the proliferative cell cycle.

Implication of cell kinetic changes during the progression of human prostatic cancer.

The daily percentage of cells proliferating and dying were determined for normal, premalignant, and cancerous prostatic cells within the prostate as well as for prostatic cancer cells in lymph node, soft tissue, and bone metastases from untreated and hormonally failing patients. These data demonstrate that normal prostatic glandular cells have an extremely low but balanced rate of cell proliferation and death (i.e., both <0.20%/day). This results in a steady-state, self-renewing condition in which there is no net growth, although the glandular cells are continuously being replaced (i.e., turnover) every 500 +/- 79 days. Transformation of these cells into high-grade prostatic intraepithelial neoplastic cells initially involves an unbalanced increase in the daily percentage of cells proliferating versus dying, such that net continuous growth occurs (i.e., mean doubling time, 154 +/- 22 days). As these early proliferation lesions continue to grow into late stage high-grade prostatic intraepithelial neoplastic cells, the daily percentage of cells dying increases further to a point equaling the daily percentage of proliferation. This results in cessation of net growth while inducing a 6-fold increase in the turnover time of these cells (i.e., 56 +/- 12 days), increasing their risk of further genetic changes. The transition of late stage high-grade prostatic intraepithelial neoplastic cells into localized prostatic cancer cells involves no further increase in proliferation but a decrease in death resulting in net continuous growth of localized prostatic cancers with a mean doubling time of >/=475 days. As compared to localized prostatic cancer cells, metastatic prostatic cancer cells within lymph nodes or bones of untreated patients have an increase in daily rate of proliferation coupled with a reduction in their daily percentage of cell death, producing net growth rates with a mean doubling time of 33 +/- 4 days and 54 +/- 5 days, respectively. Remarkably, there is no further increase in proliferation in hormonally failing patients, but instead an increase in the daily percentage of androgen-independent prostatic cancer cells dying within soft tissue or bone metastases. These changes result in doubling times which are two to three times longer (i.e., 126 +/- 21 and 94 +/- 15 days) in these lymph node and bone metastatic sites, respectively, compared to similar sites in hormonally untreated patients. These data demonstrate that the daily percentage of proliferation for either androgen-dependent or -independent metastatic prostatic cancer cells is remarkably low (i.e., <3. 0%/day), consistent with why antiproliferative chemotherapy has been of such limited value against such metastatic cells. These results also suggest that prostatic carcinogenesis starts in the second to third decade of life and may require over 50 years for progression to pathologically detectable metastatic disease.

Thromboembolic complications following radical retropubic prostatectomy. Influence of external sequential pneumatic compression devices.

The influence of external sequential compression devices (SCD) on the development of postoperative thromboembolic events was studied in 1,300 consecutive men undergoing radical retropubic prostatectomy. Of the 784 men whose perioperative management did not involve the SCD, in 9 (1.1%) thromboembolic complications developed: 7 (0.9%) pulmonary emboli (PE), and 2 (0.3%) deep venous thrombosis (DVT). In the 516 patients with SCD prophylaxis there were 12 (2.3%) thromboembolic complications: 9 (1.7%) PE, and 3 (0.6%) DVT. In patients with SCD prophylaxis, the delay from the time of surgery to the onset of thromboembolic symptoms averaged 20 +/- 12 days, and all but 1 patient suffered the complication while an outpatient; this was significantly longer than in men without SCD (11 +/- 5 days; p < 0.05). This delay in thromboembolic events was the only benefit we could demonstrate with SCD. Recognizing that most thromboembolic complications occur after discharge, new strategies for prophylaxis may be needed during this period, and patients should be well informed about the signs and symptoms of PE and DVT to avoid a delay in diagnosis and treatment.

Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis.

To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.

Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector.

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.

Phosphorylation of RNA polymerase by the murine homologue of the cell-cycle control protein cdc2.

Actively transcribing eukaryotic RNA polymerase II is highly phosphorylated on its repetitive carboxyl-terminal domain. We have isolated a protein kinase that phosphorylates serine residues in this repetitive domain. A component of this kinase is cdc2, the product of a cell-cycle control gene previously shown to be a component of M-phase-promoting factor and M-phase-specific histone H1 kinase. This observation suggests a role for the cdc2 protein kinase in transcriptional regulation.

Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase II.

The genomic locus (RPII215) encoding the largest subunit of mouse RNA polymerase II has been cloned by low stringency hybridization to a Drosophila RPII215 probe. The mouse gene consists of 28 exons which span 30 kilobases. Analysis of the nucleotide and predicted protein sequences indicates that the protein is comprised of two domains. There is a 1500 residue amino-terminal domain which contains seven regions strikingly similar to those in the beta' subunit of Escherichia coli RNA polymerase, and a carboxyl-terminal domain comprised of 52 repeats of a 7-amino-acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Among the seven highly conserved regions are a strongly basic domain consistent with a DNA-binding site and a consensus sequence characteristic of a potential zinc-binding domain. The 5' upstream region contains three tandem sequences similar to binding sites for the transcription factor SP1. Two of the introns in this gene splice at donor GC dinucleotides as opposed to previously described invariant GT sites. The identification of regions which are highly conserved as compared with bacterial and yeast RNA polymerase and other regions which are unique to the mouse protein suggests which domains of RNA polymerase large subunits are involved in aspects of transcription common to both procaryotes and eucaryotes and which are characteristic of transcription in higher organisms.