Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits.

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex. To understand fully the mechanism by which these large protein complexes perform their functions in the cell, it is crucial that all the subunits of these complexes are identified. In an attempt to identify new subunits associated with SAGA and Swi/Snf, we used tandem affinity purification, followed by a multidimensional protein identification technology to analyse the subunit composition. Our analysis identified two novel proteins, one associated with SAGA, YPL047W (Sgf11), and another associated with Swi/Snf, Rtt102.

Chromosomal localization of three vacuolar-H+ -ATPase 16 kDa subunit (ATP6V0C) genes in the murine genome.

Vacuolar-H(+)-ATPase (V-H-ATPase) is a large multimeric protein composed of at least 12 distinct subunits. The 16-kDa hydrophobic proteolipid subunit (ATP6V0C; ATPase, H(+ )transporting, lysosomal 16 kDa, V0 subunit C) plays a central role in H(+) transport across cellular membranes. We have mapped three ATP6V0C genes (Atp6v0c, Atp6v0c-ps1 and Atp6voc-ps2) in the murine genome. Atp6v0c-ps1 and Atp6v0c-ps2 map to Chromosomes 7 and 6, respectively. Atp6v0c maps to Chromosome 17, closely linked to the Tsc2 locus and D17Mit55. This region of Chromosome 17 in mouse is homologous with chromosome 16 in human where the ATP6V0C gene is localized.

Interstitial cystitis in men.

OBJECTIVES: To determine the personal characteristics, the mode of presentation, the duration of the delay in diagnosis, the number of misdiagnoses, the means to achieve diagnosis, and previous treatment provided for a group of men with interstitial cystitis (IC). METHODS: A chart review of 29 men diagnosed with IC at our facility from 1988 to 1996 was performed. Basic demographic data, historical information, laboratory findings, and endoscopic and biopsy results were tabulated. RESULTS: IC in this series of men was diagnosed at a mean age of 67.3 years. There was approximately a 4-year diagnostic lag between presentation and diagnosis. The most common prior erroneous diagnoses were prostatitis in 48% and benign prostatic hypertrophy (BPH) in 38% of the men. Ulcers were encountered cystoscopically in about 70% and biopsy specimens uniformly showed nonspecific chronic cystitis at the time of diagnosis. CONCLUSIONS: IC should be considered in the differential diagnosis of voiding disorders accompanied by irritative symptoms and pelvic pain in older men. The diagnosis should be especially considered in men who are refractory to the usual treatments for BPH and prostatitis. Cystoscopy and bladder distention under anesthesia provided the most useful objective information in our hands. Biopsy is useful to rule out inflammatory cancer but adds little to the diagnosis of IC.

Comparison of the modified vest and the direct anastomosis for radical retropubic prostatectomy.

OBJECTIVES: This retrospective study was undertaken to compare the efficacy of the Vest and direct vesicourethral anastomosis for radical prostatectomy. METHODS: Five hundred six patients who underwent consecutive radical prostatectomies at our institution were analyzed. Two hundred fifty-nine patients underwent vesicourethral anastomosis using the Vest technique and 247 underwent a direct suture anastomosis. The groups were analyzed relative to time until healing, the occurrence of anastomotic strictures, and the continence rate 1 year after surgery. RESULTS: Approximately twice as many patients who underwent the Vest procedure experienced delayed healing and 8.5% developed anastomotic strictures compared with 1.2% of the direct anastomosis group. The Vest group experienced slightly better urinary continence 1 year postoperatively. CONCLUSIONS: The Vest procedure is a reasonable alternative to direct anastomosis for radical prostatectomy and provides similar results. We suggest specific circumstances when the Vest anastomosis may be particularly useful.

The use of radiotherapy for patients with isolated elevation of serum prostate specific antigen following radical prostatectomy.

PURPOSE: An analysis was performed to assess the outcome of patients who received radiotherapy for isolated elevation of serum prostate specific antigen (PSA) levels following radical retropubic prostatectomy. MATERIALS AND METHODS: Forty-six patients were initially treated for localized prostate cancer with radical retropubic prostatectomy following negative pelvic lymphadenectomy. These patients had detectable serum PSA 6 or more months postoperatively. No patient had other clinical evidence of recurrent disease as determined by history, physical examination, bone scan, computerized tomography of the abdomen and pelvis, chest radiographs, complete blood cell counts and serum chemistry profiles. The patients received prostate bed irradiation using 10 MV. x-rays and a 4-field approach. Doses ranged from 60.0 to 67.0 Gy. in 1.8 to 2.0 Gy. fractions. Freedom from failure after radiotherapy was defined as maintaining a PSA of 0.3 ng./ml. or less without hormonal intervention. RESULTS: In 27 of the 46 patients (59%) PSA had decreased to 0.3 ng./ml. or less at last measurement without hormonal intervention. The freedom from failure rate was 50% at 3 and 5 years. More favorable responses to salvage radiotherapy occurred in patients with low grade tumors and serum PSA 1.1 ng./ml. or less at initiation of radiotherapy. Patients, receiving radiation doses of 64 Gy. or more had more favorable response rates than those receiving lesser doses. CONCLUSIONS: Isolated elevations of serum PSA following prostatectomy reflect residual disease. Radiotherapy administered to the prostate bed effectively decreased serum PSA in approximately half of the cases. This effect appears to be accomplished by eradicating tumor cells in the prostate bed.

Detection of residual prostate cancer after radical prostatectomy with the Abbott IMx PSA assay.

OBJECTIVES: This analysis was performed to define the level of serum prostate-specific antigen (PSA) measured with the Abbott IMx assay that indicates residual or progressive prostate cancer after radical retropubic prostatectomy (RRP). METHODS: Since March 1992, we have used the Abbott IMx assay to determine PSA levels. Between March 1992 and June 1994, 102 of those patients having RRPs were found to have pathologic Stage C prostate cancer. Fifty-one of these patients had at least one serum PSA measurement of 0.1 ng/mL or greater. Eight patients were excluded from the analysis because they received postoperative radiotherapy that might have influenced subsequent PSA levels. The remaining 43 patients are the subjects of this analysis and were evaluated to determine the "clinical threshold" or minimal serum PSA level after RRP indicative of progressive disease. Patients were followed for 6 to 36 months (median 23 months) from the date of the RRP. Failure was defined as a subsequent increase of PSA to greater than 0.3 ng/mL. Freedom from failure was determined using the Kaplan-Meier product limit method. RESULTS: Of the patients with at least one postoperative serum PSA level of 0.1 ng/mL, the subsequent freedom from failure was 80% at 23 months as compared with 13% in patients with at least one postoperative PSA level of 0.2 ng/mL (P = 0.003). CONCLUSIONS: Following RRP for pathologic Stage C prostate cancer, a solitary PSA level of 0.1 ng/mL (measured with the IMx assay) was followed by a progressive rise in PSA levels in only a minority of patients within the first 2 years after surgery. In contrast, the majority of patients with a postoperative PSA level of 0.2 ng/mL subsequently had progressively rising PSA levels. This indicates that a serum PSA level of 0.2 ng/mL is reflective of residual prostate cancer.

The result of radical retropubic prostatectomy and adjuvant therapy for pathologic stage C prostate cancer.

PURPOSE: The results of therapy in 288 men with pathologic Stage C prostate cancer who underwent radical retropubic prostatectomy (RRP) were analyzed to determine the effects of adjuvant therapy. METHODS AND MATERIALS: Twenty-seven of the 288 patients received preoperative neoadjuvant hormonal therapy (leuprolide acetate). Postoperatively, 60 patients received adjuvant radiotherapy (RT) to the prostate bed. Follow-up ranged from 3 to 83 months (median = 32 months). Freedom from failure (FFF) was defined as maintaining a serum PSA level of < or = 0.3 ng/ml. RESULTS: The FFF was 61% at 3 years and 45% at 5 years for the entire group. The FFF following RRP plus RT was 75% at 3 years and 57% at 5 years as compared to 56% at 3 years and 40% at 5 years for RRP without RT (p=0.049). The FFF following RRP plus neoadjuvant hormonal therapy was 58% at 3 years and 40% at 5 years as compared to 60% at 3 years and 45% at 5 years following RRP without hormonal therapy (p=0.3). In patients without seminal vesicle (SV) invasion, the FFF was 81% at 3 years and 5 years for RRP plus RT as compared to 61% at 3 years and 50% at 5 years for RRP without RT (p=0.01). In patients with SV invasion, the FFF was 61% at 3 years and 36% at 5 years for RRP plus RT as compared to 44% at 3 years and 23% at 5 years for RRP without RT (p=0.23). The projected local control rate was 83% at 5 years for those with RRP alone as compared to 100% for RRP plus RT (p=0.02). Survival at 5 years was projected to be 92% and was not significantly altered by the administration of adjuvant therapies. CONCLUSIONS: Postoperative RT was associated with significantly improved local control and FFF rates, especially in patients with tumors which did not involve the seminal vesicles.

Urinary tract stones in pregnancy.

The presence of stones during an otherwise uneventful pregnancy is a dramatic and potentially serious issue for the mother, the fetus, and the treating physicians alike. The incidence and predisposing factors are generally the same as in nonpregnant, sexually active, childbearing women. Unique metabolic effects in pregnancy such as hyperuricuria and hypercalciuria, changes in inhibitors of lithiasis formation, stasis, relative dehydration, and the presence of infection all have an impact on stone formation. The anatomic changes and physiologic hydronephrosis of pregnancy make the diagnosis and treatment more challenging. Presenting signs and symptoms include colic, flank pain, hematuria, urinary tract infection, irritative voiding, fever, premature onset or cessation of labor, and pre-eclampsia. The initial evaluation and treatment are again similar to those used for the nonpregnant population. The most appropriate first-line test is renal ultrasonography, which may, by itself, allow the diagnosis to be made and provide enough information for treatment. Radiographic studies, including an appropriately performed excretory urogram, give specific information as to size and location of the stones, location of the kidneys, and differential renal function and can be used safely, but the ionizing radiation risks should be considered. All forms of treatment with the exception of extracorporeal shock wave lithotripsy and some medical procedures are appropriate in the pregnant patient. Close coordination by the urologist, the obstetrician, the pediatrician, the anesthesiologist, and the radiologist is required for the appropriate care of these patients.

Assessment of bladder function after lumbar decompressive laminectomy for spinal stenosis: a prospective study.

Lumbar spinal stenosis is a common problem in elderly patients. In its more advanced forms, it typically causes intractable leg pain, but many patients also manifest varying degrees of bladder dysfunction. The goal of lumbar decompressive laminectomy is relief of leg pain and paresthesias, yet some patients also achieve improvement in bladder function. This study prospectively investigated patients with lumbar spinal stenosis to determine whether laminectomy had any effect on urological function. Of the 20 patients in the study, 10 were men and 10 women (average age 70.9 years). All patients had severe lumbar stenosis affecting between two and four spinal segments, and all reported some degree of bladder dysfunction. Cystoscopy and urodynamic testing were completed preoperatively. A standard decompressive laminectomy was performed over the appropriate number of spinal segments. Urodynamic studies were repeated at 2 and 6 months postoperatively. At the 6-month follow-up review, bladder function was subjectively improved in 12 patients (60%) and unchanged in eight (40%). Postvoiding residual urine volume was the urodynamic factor most likely to be improved by laminectomy. In nine patients (45%), baseline postvoiding residual urine volume was elevated and all nine had improvement postoperatively. In the remaining 11 patients (55%), this urine volume was normal before and after surgery. Maximum urine flow rates also improved, but the results of cytometrography and electromyography, urine flow pattern, and bladder capacity were unchanged postoperatively. Cystoscopy detected previously undiagnosed malignancy of the lower urinary tract in two patients (10%). It is concluded that lumbar decompressive laminectomy can have a beneficial effect on bladder function in a significant number of patients with advanced lumbar spinal stenosis.

Inhibition of calcineurin by cyclosporin A-cyclophilin requires calcineurin B.

The interaction of the immunosuppressive complex cyclosporin A-cyclophilin (CsA-CyP) with the Ca2+/calmodulin-dependent protein phosphatase calcineurin is investigated using a recombinant form of the A subunit of calcineurin (rCNA). Only in the presence of purified calcineurin B (CNB) does rCNA show the response of native calcineurin, i.e. 50% inhibition of rCNA phosphatase activity at 6 nM human cyclophilin B and 0.6 microM human cyclophilin A using [32P]casein as substrate, yet stimulation of activity with p-nitrophenyl phosphate as substrate. This study demonstrates that the B subunit is necessary to confer sensitivity of calcineurin to CsA-CyP.

Clinical trials of the Northgate SD-3 dual-purpose lithotriptor for renal calculi.

The Northgate SD-3 is a bathless, portable shock wave lithotriptor made in the United States. It uses ultrasound localization and spark-gap, electrode-generated shock waves to fragment calculi in the upper urinary tract. Since October 1987, 312 treatments have been performed on 281 patients (286 kidneys) with stone burdens less than 2 cm. during clinical trials at 6 investigational sites in the United States. A fragmentation rate of 94% was achieved. Of the treatments 78% were judged successful (stone-free or fragments of less than 5 mm. remaining in an asymptomatic patient) and a 3-month stone-free rate of 58% was noted. The retreatment rate was 9% and the ancillary procedure rate was 5%. The complications (hematuria, ecchymosis, pain, obstruction) were mild and not unlike those seen in patients undergoing lithotripsy with other devices.

Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B.

The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.

Excretory urography during extracorporeal shock-wave lithotripsy: a localization alternative.

An alternative localization technique or extracorporeal shock wave lithotripsy using devices that use fluoroscopic targeting is presented. Excretory urography during lithotripsy can provide valuable targeting information and the results of treatment in a manner that may prove useful in the treatment of urinary calculi.

Direct sequencing from touch preparations of human carcinomas: analysis of p53 mutations in breast carcinomas.

A new technique for characterizing somatic mutations in very small samples of cellularly heterogeneous human cancer tissue was developed and tested using mutations in the p53 gene in breast carcinomas as a model system. The technique combines touch preparation of specimens to obtain homogeneous clusters of carcinoma cells free of normal cells with a nested pair of polymerase chain reaction (PCR) amplifications of DNA to increase the amount of target gene sequence sufficiently to permit direct sequencing of the p53 gene. Touch preparations of fresh or previously frozen tissue from human adenocarcinomas derived from several organs were stained, and clusters of 10-50 malignant cells were transferred by pipette into microfuge tubes for PCR amplification. Exons 5-9 of the p53 gene, which contain the major mutational hot spots associated with most human cancers, were sequenced by the following steps: 1) two rounds of PCR amplification using DNA Taq polymerase and two sets of oligonucleotide primers, the second set being nested within the segment amplified by the first set and having attached T7 and SP6 phage promoter sequences, 2) transcription of the amplified DNA sequences with T7 and SP6 RNA polymerases, and 3) dideoxy sequencing of single-stranded RNA transcripts with reverse transcriptase and with additional oligonucleotide primers to achieve specificity for this unique region of the genome. The utility of this approach is illustrated by our success in detecting and analyzing point mutations in cell clusters from four of 11 primary adenocarcinomas of the human breast.

Characterization of Vero cells.

A cell line used in the production of biologicals should be free of infectious agents, and 'described with respect to cytogenetic characteristics and tumorigenicity'. Vero, a continuous cell line derived from a normal African green monkey kidney, was examined for the presence of retroviruses and for tumorigenic potential. We were unable to detect the presence of retroviruses by reverse transcriptase assay, electron microscopy or hybridization of cellular genomic DNA with Mason-Pfizer monkey virus DNA probes. In addition, passage 156 Vero cells did not form progressively growing tumors in nude mice or grow with high efficiency in soft agarose.

Several classes of retroviruses are produced by an AKR mouse T lymphoma cell line.

Characterization of the viruses produced by the spontaneous T lymphoma cell line SL3 is presented. Using supernatant fluids or direct co-cultivation of cells, the SL3 cell line was found to produce replication-defective viruses in excess of replication-competent viruses. The replication-competent viruses released were predominantly those negative in the XC plaque assay (XC-); XC+ viruses represented a minor population. However, when the SL3-derived viruses were passed in mouse embryo fibroblasts, XC- viruses were rarely recovered, and XC+ viruses were readily isolated. These viruses were all ecotropic and lymphomagenic. Viruses with dual host range and non-oncogenic ecotropic viruses were not isolated from the lymphoma cells. Two replication-defective viruses from SL3 cells were studied. Both could be rescued by non-oncogenic retroviruses and were then lymphomagenic. One defective virus appeared related to XC+ viruses. In these studies, the XC+ and XC- viruses appeared to represent two different interference classes using separate cell receptors. Taken together, these experiments show that the SL3 T lymphoma cells replicate a variety of viruses most of which are lymphomagenic. Virus replication and/or virus integration may be the means of maintaining the malignant phenotype of these T lymphoma cells.