The pitfalls of treating anorectal conditions after radiotherapy for prostate cancer.

We present a salutary lesson learned from three cases with significant complications that followed anorectal intervention in the presence of radiation proctitis due to prior radiotherapy for adenocarcinoma of the prostate. After apparent routine rubber band ligation for painful haemorrhoids, one patient developed a colo-cutaneous fistula. Following laser coagulation for radiation proctitis, one patient required a pelvic exenteration for a fistula, while another developed a rectal stenosis. Those diagnosing and treating colonic conditions should be mindful of the increased prevalence of patients who have had radiotherapy for prostate cancer and the potential for complications in treating these patients.

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA.

Cell divisions that produce progeny differing in their patterns of gene expression are key to the development of multicellular organisms. In the budding yeast Saccharomyces cerevisiae, mother cells but not daughter cells can switch mating type because they selectively express the HO endonuclease gene. This asymmetry is due to the preferential accumulation of an unstable transcriptional repressor protein, Ash1p, in daughter cell nuclei. Here it is shown that ASH1 messenger RNA (mRNA) preferentially accumulates in daughter cells by a process that is dependent on actin and myosin. A cis-acting element in the 3'-untranslated region of ASH1 mRNA is sufficient to localize a chimeric RNA to daughter cells. These results suggest that localization of mRNA may have been an early property of the eukaryotic lineage.

Current concepts in female stress urinary incontinence.

Urinary incontinence is a social burden for up to one-third of the adult female population. Careful assessment, a methodical approach and appropriate treatment can lead to long-term success in the management of these patients. This article gives an outline of current concepts in the evaluation and treatment of stress urinary incontinence.

Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo.

The sorting of the Ash1 protein to the daughter nucleus of Saccharomyces cerevisiae in late anaphase of the budding cycle correlates with the localization of ASH1 mRNA at the bud tip [1] [2]. Although the 3' untranslated region (3' UTR) of ASH1 is sufficient to localize a reporter mRNA, it is not necessary, a result which indicates that other sequences are involved [1]. We report the identification of three additional cis-acting elements in the coding region. Each element alone, when fused to a lacZ reporter gene, was sufficient for the localization of the lacZ mRNA reporter to the bud. A fine-structure analysis of the 3' UTR element showed that its function in mRNA localization did not depend on a specific sequence but on the secondary and tertiary structure of a minimal 118 nucleotide stem-loop. Mutations in the stem-loop that affect the localization of the lacZ mRNA reporter also affected the formation of the localization particles, in living cells, composed of a green fluorescent protein (GFP) complexed with lacZ-ASH1-3' UTR mRNA [3]. A specific stem-loop in the 3' UTR of the ASH1 mRNA is therefore required for both localization and particle formation, suggesting that complex formation is part of the localization mechanism. An analysis on one of the coding-region elements revealed a comparable stem-loop structure with similar functional requirements.

Planning for a national effort to enable and accelerate discoveries in pharmacogenetics: the NIH Pharmacogenetics Research Network.

The Pharmacogenetics Research Network (PGRN) was conceived as a broad network-based approach to research studies in pharmacogenetics and pharmacogenomics, with the central feature of a database that would be hypothesis-generating and open to all scientific investigators. The original working group emphasized the importance of carefully relating phenotypes for drug responses to genotypes, and understanding the relationships functionally and mechanistically. The mission of the PGRN is to advance our knowledge of the genetic basis for variable drug responses. The ultimate goal of the effort is to determine clinically significant sequence variations in order to predict the right choice and dose of medications for individuals and to prepare for implementation of this information to improve health care.

GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator.

The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.

SmgGDS is a transient nucleolar protein that protects cells from nucleolar stress and promotes the cell cycle by regulating DREAM complex gene expression.

The chaperone protein and guanine nucleotide exchange factor SmgGDS (RAP1GDS1) is a key promoter of cancer cell proliferation and tumorigenesis. SmgGDS undergoes nucleocytoplasmic shuttling, suggesting that it has both cytoplasmic and nuclear functions that promote cancer. Previous studies indicate that SmgGDS binds cytoplasmic small GTPases and promotes their trafficking to the plasma membrane. In contrast, little is known about the functions of SmgGDS in the nucleus, or how these nuclear functions might benefit cancer cells. Here we show unique nuclear localization and regulation of gene transcription pathways by SmgGDS. Strikingly, SmgGDS depletion significantly reduces expression of over 600 gene products that are targets of the DREAM complex, which is a transcription factor complex that regulates expression of proteins controlling the cell cycle. The cell cycle regulators E2F1, MYC, MYBL2 (B-Myb) and FOXM1 are among the DREAM targets that are diminished by SmgGDS depletion. E2F1 is well known to promote G1 cell cycle progression, and the loss of E2F1 in SmgGDS-depleted cells provides an explanation for previous reports that SmgGDS depletion characteristically causes a G1 cell cycle arrest. We show that SmgGDS localizes in nucleoli, and that RNAi-mediated depletion of SmgGDS in cancer cells disrupts nucleolar morphology, signifying nucleolar stress. We show that nucleolar SmgGDS interacts with the RNA polymerase I transcription factor upstream binding factor (UBF). The RNAi-mediated depletion of UBF diminishes nucleolar localization of SmgGDS and promotes proteasome-mediated degradation of SmgGDS, indicating that nucleolar sequestration of SmgGDS by UBF stabilizes SmgGDS protein. The ability of SmgGDS to interact with UBF and localize in the nucleolus is diminished by expressing DiRas1 or DiRas2, which are small GTPases that bind SmgGDS and act as tumor suppressors. Taken together, our results support a novel nuclear role for SmgGDS in protecting malignant cells from nucleolar stress, thus promoting cell cycle progression and tumorigenesis.

Emphysematous cystitis in an elderly patient with ulcerative colitis.

Emphysematous cystitis is a rare complication of lower urinary tract infections. The disease is characterized by air within the bladder wall and lumen and commonly occurs in middle-aged diabetic women. Intramural bladder gas seen on imaging is pathognomonic for this condition. The severity of the illness varies widely from cases diagnosed incidentally to patients presenting with life-threatening sepsis. We report the case of an 80-year-old non-diabetic man presenting with emphysematous cystitis after a total colectomy for ulcerative colitis.

SIP1 is a catabolite repression-specific negative regulator of GAL gene expression.

The yeast Snf1p kinase is required for normal expression of many genes involved in utilization of non-glucose carbon. Snf1p is known to associate with several proteins. One is Sip1p, a protein that becomes phosphorylated in the presence of Snf1p and thus is a candidate Snf1p kinase substrate. We have isolated the SIP1 gene as a multicopy suppressor of the gal83-associated defect in glucose repression of GAL gene expression. Multicopy SIP1 also suppressed the gal82-associated defect in glucose repression, suggesting that SIP1, GAL83 and GAL82 function interdependently. Multicopy SIP1 gene reduces GAL1, GAL2, GAL7 and GAL10 gene expression three- to fourfold in cells grown in the presence of glucose but has no effect in cells grown on nonrepressing carbon. Sip1-deletion cells exhibited a two- to threefold increase in GAL gene expression compared to wild-type cells when grown on glucose. These studies show that SIP1 is a catabolite repression-specific negative regulator of GAL gene expression. Northern analysis revealed two SIP1 transcripts whose relative abundance changed with carbon source. Western blots revealed that Sip1p abundance is not markedly affected by carbon source, suggesting that Sip1p may be regulated post-translationally.

Ureteral endometriosis and ovarian mucinous cystadenoma presenting with acute pyonephrosis.

BACKGROUND: Endometriosis is a common disease, but ureteral involvement is rare. Nonspecific clinical presentations of ureteral endometriosis may result in diagnostic difficulty. AIM: To discuss the diagnosis and management of such a case. METHODS: To report a case of ureteral involvement with endometriosis and review the literature. RESULTS: The case presented with right lower quadrant pain giving rise to initial diagnostic possibility of acute appendicitis. Subsequent evaluation revealed the diagnosis of right pyonephrosis due to midureteral endometriosis with right ovarian mucinous cystadenoma. CONCLUSION: The diagnosis of ureteral endometriosis requires a high index of clinical suspicion. The importance of ultrasound in the evaluation of acute abdomen in women can not be overemphasised.

A 30-year experience of Millin's retropubic prostatectomy: Has this classic operation derived by a President of the College in Ireland stood the test of time?

INTRODUCTION: In patients with large gland volume, open prostatectomy/adenoma enucleation remains a valuable surgical option in treating large obstructing prostates. We report our series of open prostatectomies spanning 32 years from a single institution. PATIENTS AND METHODS: We retrospectively reviewed all patients who underwent open prostatectomy between 1980 and 2012. Patient demographical, clinical, pre- and postoperative data and final histology were retrieved from hospital in-patient enquiry system and chart review. RESULTS: A total of 161 patients underwent Millin's prostatectomy by seven surgeons between 1980 and 2012. The mean blood loss was 1,381 mls (range 300-3,675 mls). One-third (34%) of patients (n = 55) received a blood transfusion. The mean weight of prostate tissue removed was 119 g (median 112 g, range 17-372 g). 6.6 % of pathological specimens revealed incidental prostate cancer, of which 78% were well differentiated (Gleason score ≤ 6). The mean weight of prostate tissue removed in patients who received a transfusion was 124 g. Trial of micturition (TOM) was performed at a mean of 9 days (median 9 days, range 5-25 days) with 94% of patients having a successful trial of voiding. 6% of cases early in the series failed to void initially, but did so at later removal of catheter while still in hospital. 45 patients (28%) of patients developed peri- or postoperative complications. There were three deaths (1.9%). CONCLUSION: Open Millin's prostatectomy popularized over half a century ago continues to be a valuable option for the surgical treatment of high-volume prostate glands with excellent outcomes for patients.

Agonist-induced down-regulation of m1 muscarinic receptors and reduction of their mRNA level in a transfected cell line.

Agonist-induced reduction in both the number of m1 muscarinic receptors and the mRNA coding for the receptor protein was investigated in Chinese hamster ovary (CHO) cells which were transfected with the m1 muscarinic receptor gene. Receptor concentration was measured by the specific binding of the muscarinic ligand, [3H]quinuclidinyl benzilate ([3H]QNB), and Northern blot hybridization analysis was used to evaluate the levels of receptor mRNA. Incubation of cells with 1 mM of the muscarinic agonist, carbamylcholine (CBC), for 24 h decreased receptor density and mRNA levels in cells by 65% and 73%, respectively. These results indicate that agonist-induced down-regulation of m1 muscarinic receptors might be due to, at least in part, a decrease in receptor synthesis resulting from a reduction in the steady-state level of their mRNA.

Cytosolic calcium after carbon tetrachloride, 1,1-dichloroethylene, and phenylephrine exposure. Studies in rat hepatocytes with phosphorylase a and quin2.

Carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE), both hepatotoxins, inhibit sequestration of Ca2+ by rat liver endoplasmic reticulum (ER) both in vivo and in vitro. It is possible that, as a result, cytosolic Ca2+ concentrations rise in liver cells. In experiments presented here, isolated hepatocytes were exposed to CCl4, DCE, and phenylephrine (PE), a non-hepatotoxic alpha 1-adrenergic agent that mobilizes Ca2+. Cytoplasmic Ca2+ concentrations were evaluated by two methods: indirectly by assaying the activity of glycogen phosphorylase a, and directly by monitoring the fluorescence of quin2. In primary hepatocyte cultures, CCl4, DCE, and PE exposure increased the activity of phosphorylase a at 5 min from 39 +/- 2 to 130 +/- 12, 80 +/- 13, and 97 +/- 10 nmoles PO4(3-)/mg protein/min respectively. In rat hepatocyte suspensions loaded with quin2 and exposed to CCl4, DCE, or PE, cytosolic Ca2+ concentrations were elevated within 20 sec to 0.83 +/- 0.13, 0.59 +/- 0.06 and 0.99 +/- 0.14 microM Ca2+ respectively. Basal Ca2+ levels in these cells averaged 0.25 +/- 0.03 microM. Thus, CCl4 and PE apparently increased cytosolic Ca2+ levels to approximately the same extent, whereas DCE was somewhat less effective. The durations of the effects of CCl4 and PE were examined further by determining their time courses of elevated phosphorylase a activity. In hepatocyte cultures, increased phosphorylase a activity persisted through at least 60 min following CCl4 exposure. In contrast, phosphorylase a activity returned to basal levels by 20 min after PE. Increases in cytoplasmic Ca2+ levels that are sustained rather than transient may distinguish these hepatotoxic chlorinated aliphatic hydrocarbons from non-toxic hormonal agents.

Inhibition of liver endoplasmic reticulum calcium pump by CCl4 and release of a sequestered calcium pool.

One of the earliest effects observed in rat liver after CCl4 administration is inhibition of an ATP-dependent calcium pump found at the endoplasmic reticulum. This report confirms that the amount of calcium associated with the microsomal fraction is reduced after CCl4 administration and, for the first time, demonstrates time-, dose-, and metabolism-dependent relationships between inhibition of the liver microsomal calcium pump and the amount of calcium found in the microsomal fraction. Furthermore, release of calcium from the endoplasmic reticulum is shown to cause activation of a cytoplasmic enzyme that responds to increases of ionized calcium, glycogen phosphorylase. This suggests that the endoplasmic reticulum calcium pump sequesters an intracellular pool of calcium within the endoplasmic reticulum. This pool of calcium may be released into the cytoplasm as a consequence of inhibition of the calcium pump by CCl4.

Impact of halogenated compounds on calcium homeostasis in hepatocytes.

Halocarbons (CCl4, 1,1-dichlorethylene) cause a wide spectrum of effects and injury in hepatocytes. One early effect of these compounds is the inhibition and destruction of the endoplasmic reticulum (ER) calcium pump. Subsequent to inhibition of this pump, the ER calcium pool is depleted and cytosolic levels of calcium are increased for a prolonged period of time. This effect of halocarbons has been characterized and is similar in vivo and in vitro. The importance of this redistribution of cell calcium in expression of halocarbon injury of hepatocytes has not been fully resolved. Several degradative enzymes (phospholipases, proteases) have been implicated as calcium-dependent mediators in toxicity. Our preliminary studies of the effect of calcium redistribution suggest that activation of a calcium-sensitive endonuclease in liver does not play a central role in initiating the lethal effect of halocarbons on hepatocytes.

Cloning and sequencing of a human liver carboxylesterase isoenzyme.

A human liver lambda gt11 library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxylesterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats.

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Chair rise strategies in the elderly.

The elderly often have difficulty with rising from a chair. The purpose of this study was to characterize their rising strategies. A group of 22 elderly adults with a range of functional impairments was asked to rise from chairs of varying heights. Videotape motion analysis was used to identify strategies, estimate centre of mass, and measure time to rise. Three movement strategies were identified, "momentum transfer", "stabilization", and "combined" based on the velocity of trunk movement and base of support rearrangement. "Momentum transfer" uses horizontal momentum developed in the trunk to rise; "stabilization" uses centre of mass and base of support repositioning but very little momentum; "combined" uses elements of both momentum transfer and stabilization. Differences in the time to rise and the centre of mass to base of support separation between the momentum transfer and stabilization strategies were significant at each chair height. The momentum transfer, combined, and stabilization may form a continuum of chair rise strategies.