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R L Hackett

Publications and source records attributed to R L Hackett.

At least 19 recordsLinked to original sources
PubMed2000

The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

The Ess1/Pin1 peptidyl-prolyl isomerase (PPIase) is thought to control mitosis by binding to cell cycle regulatory proteins and altering their activity. Here we isolate temperature-sensitive ess1 mutants and identify six multicopy suppressors that rescue their mitotic-lethal phenotype. None are cell cycle regulators. Instead, five encode proteins involved in transcription that bind DNA, modify chromatin structure or are regulatory subunits of RNA polymerase II. A sixth suppressor, cyclophilin A, is a member of a distinct family of PPIases that are targets of immuno suppressive drugs. We show that the expression of some but not all genes is decreased in ess1 mutants, and that Ess1 interacts with the C-terminal domain (CTD) of RNA polymerase II in vitro and in vivo. The results forge a strong link between PPIases and the transcription machinery and suggest a new model for how Ess1/Pin1 controls mitosis. In this model, Ess1 binds and isomerizes the CTD of RNA polymerase II, thus altering its interaction with proteins required for transcription of essential cell cycle genes.

Amino Acid Sequence↗
PubMed2000

Free radical scavengers, catalase and superoxide dismutase provide protection from oxalate-associated injury to LLC-PK1 and MDCK cells.

PURPOSE: Current studies have provided evidence that exposure of renal epithelial cells to oxalate and calcium oxalate crystals induces lipid peroxidation and injures the cells. Since oxidant/antioxidant balance is likely to play a critical role, we determined the effect of antioxidant scavengers on production of free radicals and injury to LLC-PK1 and MDCK cells from exposure to oxalate (Ox) or Ox + calcium oxalate monohydrate (COM) crystals. MATERIALS AND METHODS: LLC-PK1 and MDCK cells were grown in monolayers and exposed to 1.0 mmol. Ox or 1.0 mmol. Ox + 500 microg. /ml. COM crystals for 120 or 240 minutes. We measured the release of lactate dehydrogenase (LDH) as a marker for cell injury and malondialdehyde (MDA) as a marker of lipid peroxidation. Superoxide and hydroxyl radicals were measured in the presence or absence of 400 U/ml. catalase, or superoxide dismutase (SOD). RESULTS: Exposure of LLC-PK1 cells to Ox resulted in a significant increase in MDA and release of LDH, which was further elevated when COM crystals were added. MDCK cells responded similarly to both challenges, but showed significantly less impact when compared with LLC-PK1 cells. Both treatments were associated with significant increase in the generation of hydroxyl and superoxide radicals by both cell types. In both cell lines, the addition of catalase or SOD significantly reduced the increase of MDA and release of LDH. CONCLUSIONS: Results of the present study indicate that both Ox and COM crystals are injurious to renal epithelial cells and the injury is associated with generation of free radicals. Cells of proximal tubular origin are more susceptible than those of distal tubules and collecting ducts. Free radical scavengers, catalase and SOD provide significant protection.

Animals↗
PubMed1999

Cells of proximal and distal tubular origin respond differently to challenges of oxalate and calcium oxalate crystals.

LLC-PK1 and Madin-Darby canine kidney (MDCK) cells were used to study the role of free radicals in renal epithelial injury during exposure to oxalate ions (Ox) and calcium oxalate monohydrate (COM) crystals. The cell cultures were exposed for 120 or 240 min to 1.0 mmol Ox or 1.0 mmol Ox plus 500 microg/ml of COM crystals averaging 1.0 microm in size. Exposure of both LLC-PK1 and MDCK cells to Ox alone increased the leakage of lactate dehydrogenase, which was further enhanced when cells were exposed to Ox + COM crystals. The release of lactate dehydrogenase from the LLC-PK1 cell line, however, was significantly higher than that from MDCK cells. LLC-PK1 cells also showed a significant increase in malondialdehyde (MDA) content on Ox challenge. MDA content was even higher when LLC-PK1 cells were challenged with Ox + COM crystals. However, in MDCK cells, the elevated MDA content was similar in both treatment groups, suggesting that these cells may be more resistant to the calcium oxalate crystals. Glutathione peroxidase activity was decreased in both LLC-PK1 and MDCK cells. Challenging cells with Ox + COM resulted in decreased catalase activity in LLC-PK1, but increased catalase activity in MDCK cells. Superoxide dismutase activity and reduced glutathione content were not significantly different in either cell type when challenged with Ox or Ox + COM. Previous in vivo animal studies yielded indirect evidence for the increased lipid peroxidation during hyperoxaluria-induced nephrolithiasis. However, in an animal model, it is difficult to separate the effect of Ox from Ox in combination with COM crystals. This study suggests that the injury to renal tubular epithelial cells is accompanied by lipid peroxidation when exposed to Ox. The injury is augmented when COM crystals are included. LLC-PK1 cells are more susceptible to Ox-associated injury than MDCK cells.

Animals↗
PubMed1999

Crystal-cell interaction and apoptosis in oxalate-associated injury of renal epithelial cells.

Two renal epithelial cell lines, LLC-PK1 and Madin-Darby canine kidney (MDCK), were grown in monolayers and exposed to oxalate (Ox) and/or calcium oxalate (CaOx) crystals to investigate cellular responses to these challenges. In addition, LLC-PK1 cells were exposed to high concentrations of Ox for various time periods to investigate the role of apoptosis in Ox-associated cell injury. Both cell types showed signs of damage when exposed to Ox. However, LLC-PK1 cells appeared more sensitive than MDCK cells. There was a significant increase in release of lactate dehydrogenase into the medium and decrease in trypan blue exclusion by cells in the monolayer. Most noticeable was the detachment of cells from the substrate. Exposure of cells to CaOx crystals resulted in their attachment to cell surfaces followed by internalization. Using flow cytometry for quantification of apoptotic cells, transmission electron microscopy for morphology, and electrophoresis for DNA laddering detection, we observed significant apoptotic changes including condensation and margination of nuclear chromatin, DNA fragmentation, and migration of phosphatidylserine of the plasma membrane from inside to the cell surface. However, these cells also showed some necrotic changes such as loss of plasma membrane integrity and release of lactate dehydrogenase, indicating that the apoptotic process was interrupted.

Animals↗
PubMed1997

Lipid peroxidation in ethylene glycol induced hyperoxaluria and calcium oxalate nephrolithiasis.

PURPOSE: To determine if lipid peroxidation plays a role in renal injury associated with experimental nephrolithiasis. MATERIALS AND METHODS: Hyperoxaluria was produced in rats by ethylene glycol in drinking water. At 15, 30 and 60 days of treatment, urinary oxalate, lipid peroxide, calcium oxalate crystals, enzymes and tissue lipid peroxide were measured. RESULTS: Urinary oxalate increased significantly at all time periods and was associated with crystalluria. Lipid peroxides in kidney tissue and urine increased at all time periods. Tissue calcium oxalate crystal deposits from 0 to 1+ were present on day 15, but present in all animals on days 30 and 60. Renal tubular cell damage was confirmed by an increase in urinary marker enzymes. CONCLUSIONS: Renal cell damage is associated with lipid peroxide production indicating cell injury due to the production of free radicals. The damage appears due primarily to hyperoxaluria and is augmented by crystal deposition in the renal tubules.

Animals↗
PubMed1996

The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally. These gene deletion results are evaluated in the context of the redundancy and degeneracy inherent in many genetic networks. Our cDNA analyses and data-base searches reveal that the predicted dodo protein has homologs in other eukaryotes and that it is made up of two different domains. The first, designated WW, is involved in protein-protein interactions and is found in functionally diverse proteins including human dystrophin. The second is involved in accelerating protein folding and unfolding and is found in Escherichia coli in a new family of peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8). In eukaryotes, PPIases occur in the nucleus and the cytoplasm and can form stable associations with transcription factors, receptors, and kinases. Given this particular combination of domains, the dodo protein may well participate in a multisubunit complex involved in the folding and activation of signaling molecules. When we expressed the dodo gene product in Saccharomyces cerevisiae, it rescued the lethal phenotype of the ESS1 cell division gene.

Amino Acid Sequence↗
PubMed1996

Bladder wall fibrosis following intravesical mitomycin treatment for superficial bladder cancer.

Mitomycin is used extensively for the prevention of recurrence of superficial bladder cancer. Most treatment regimens of mitomycin are long term, since this seems more effective in preventing recurrence. During treatment some patients develop cystitis of variable severity, which may lead to mucosal ulcerations and cessation of treatment. We report a case in which long-term treatment with mitomycin, following a single episode of transitional cell bladder cancer, has led to bladder fibrosis and loss of its function, without evidence of tumor recurrence.

Administration, Intravesical↗
PubMed1995

Rectus fascial wrap: early results of a modification of the rectus fascial sling.

We describe an operation that increases bladder neck resistance in patients with urinary incontinence. It is a modification of the rectus fascial sling that was designed as an adjunct to augmentation cystoplasty and is used in association with clean intermittent catheterization. The operation is performed by circumferentially wrapping a rectus fascial defatted free graft around the bladder neck and suturing it to appose the bladder neck. The procedure was done in 17 patients, including 10 with myelodysplasia, 3 with sacral lipoma, 3 with bladder exstrophy and 1 with nonneurogenic neurogenic bladder. Leak point pressure improved in patients in whom it was measured preoperatively and postoperatively. Complications developed in 5 patients, including difficulty with catheterization in 2, ventral hernia at the graft harvest site in 1, bladder calculus in 1 and detrusor hyperreflexia in 1. Early results with the bladder fascial wrap indicate that it has the ability to improve continence in patients with a dysfunctional bladder neck who have undergone augmentation.

Adolescent↗
PubMed1995

Alterations in MDCK and LLC-PK1 cells exposed to oxalate and calcium oxalate monohydrate crystals.

Structural analysis of human kidney stones reveals the presence of cellular membranes and other cell fragments. Experimentally, calcium oxalate crystallization is facilitated when an exogenous nephrotoxin is given with ethylene glycol, thus providing cellular degradation products to act as heterogeneous nuclei. In this report, we tested whether oxalate alone could act as a cell toxin capable of producing damaged cells without the presence of an exogenous agent. Cultured LLC-PK1 and MDCK cells, when exposed to 1.0 mmol KOx, a concentration at the limit of metastability for calcium oxalate nucleation, were severely damaged as measured by specific lactate dehydrogenase (LDH) release in the spent media and by trypan blue exclusion. This effect was magnified by the addition of pre-formed calcium oxalate monohydrate crystals; the injury was significantly amplified when compared to exposure to oxalate alone. Scanning electron microscopy studies illustrated attachment of crystals to cells with loss of cell-to-cell and cell-to-substrate contact, as cells were released from the monolayer. In both oxalate and combined crystal-oxalate studies, more cells were released from the monolayer and exhibited considerably more damage when compared to controls. Oxalate, at the limit of metastability for calcium oxalate, is a cell toxin and can produce cellular degradation products. This effect is increased significantly by the addition of calcium oxalate monohydrate crystals.

Animals↗
PubMed1994

Madin-Darby canine kidney cells are injured by exposure to oxalate and to calcium oxalate crystals.

The reaction of Madin-Darby canine kidney cells (MDCK) to potassium oxalate (KOx), calcium oxalate monohydrate (COM) crystals, or a combination of the two was studied. The most noticeable effect of exposure of the cells to either KOx or COM crystals was loss of cells from the monolayer ranging from 20% to 30%, depending upon the particular treatment. Cellular enzyme values in the media were elevated significantly by 12 h of exposure, although in specific instances, elevated levels occurred at earlier time periods. As regards the monolayer, trypan blue exclusion was decreased significantly, although amounting to only a 4-5% reduction. Specific tritiated release occurred at 4 and 12 h after exposure to KOx and at 12 h after exposure to crystals. Structurally, COM-cell interactions were complex and extensive endocytosis was noted. Cells were released from culture either as cell-crystal complexes or from the intercellular spaces after exocytosis. When treatments were combined the effects were only slightly additive, but the two treatments potentiated each other: all media enzyme levels (with one exception) were elevated at 2 h, tritiated adenine release was present at 4 h, and there was more extensive cell loss from the culture monolayer. These data suggest that both KOx and COM crystals damage MDCK cells when applied alone, and in concert they act synergistically.

Adenine↗
PubMed1993

Role of organic matrix in urinary stone formation: an ultrastructural study of crystal matrix interface of calcium oxalate monohydrate stones.

Human calcium oxalate monohydrate (COM) urinary stones were decalcified by treatment with a mixture of ethylenediaminetetraacetic acid (EDTA) solution and Karnovsky's fixative after embedding in bactoagar. Decalcified stones were examined by light microscopy, and also by scanning and transmission electron microscopy. Stones had distinct nuclei that were occupied by amorphous or apatitic calcium phosphate or aggregates of spherulitic COM crystals. EDTA insoluble matrix was ubiquitous in stones and consisted largely of finely matted fibrous material. It was organized in concentric laminations in the peripheral area of the stone but appeared highly disorganized in the stone center. Crystals were replaced by crystal ghosts. Organic matrix was present both inside the crystals and in the intercrystalline spaces. The study indicates a very close association between crystals and organic matrix. The relationship appears to begin early in crystal formation and persists throughout the formative and growth phases of the urinary stones.

Calcium Oxalate↗
PubMed1993

Magnesium oxide administration and prevention of calcium oxalate nephrolithiasis.

We studied the effect of oral administration of magnesium oxide (MgO) on calcium oxalate (CaOx) nephrolithiasis in rats. Nephrolithiasis was induced by administration of 1.0% ethylene glycol (EG) in drinking water. Magnesium oxide was given mixed with food at 500 mg./100 g. rat chow. Dispensation of MgO resulted in a significant increase of urinary pH and a modest increase in urinary excretion of citrate. Urinary excretion of oxalate started to decline by day 14 and was significantly reduced on days 21 and 28. All rats receiving EG displayed crystalluria. From the group receiving EG only, 3 of 4 rats sacrificed on day 15 and 2 of 4 rats sacrificed on day 29 had CaOx crystal deposits in their kidneys. None of the 8 rats who received both EG and MgO had CaOx nephrolithiasis. Thus our findings indicate that dispensation of magnesium as MgO can be beneficial against calcium oxalate nephrolithiasis.

Animals↗
PubMed1992

Acute hyperoxaluria, renal injury and calcium oxalate urolithiasis.

Single intraperitoneal injections of three, seven, or 10 mg. of sodium oxalate per 100 gm. of rat body weight were administered to male Sprague-Dawley rats. At various times after the injection, urine samples were analyzed for oxalate, and urinary enzymes, alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and N-acetyl-beta-glucosaminidase. The kidneys were processed for light microscopy and renal calcium and oxalate determination. Oxalate administration resulted in an increase in urinary oxalate and formation of calcium oxalate crystals in the kidneys. The amount and duration of urinary excretion of excess oxalate and retention of crystals in the kidneys correlated with the dose of sodium oxalate administered. At a low oxalate dose of three mg./100 gm., crystals moved rapidly down the nephron and cleared the kidneys. At higher doses crystals were retained in kidneys and at a dose of 10 mg./100 gm. were still there seven days post-injection. Crystal retention was associated with enhanced excretion of urinary enzymes indicating renal tubular epithelial injury.

Acute Disease↗
PubMed1992

Multiple criteria in sonographic diagnosis of prostatic carcinoma allows identification of all glands harboring clinically significant tumor: an in vitro study with histopathologic correlation.

Fresh whole prostate glands were examined systematically in a water bath to look for four sonographic criteria for prostatic carcinoma: well-circumscribed hypoechoic regions, external asymmetry of the gland, increase in anterior-posterior diameter ratio, and irregularity of the prostatic capsule. Whole mount sections were examined to correlate the histopathologic and sonographic findings. Using hypoechogenicity alone, 26 of 43 glands harboring tumor were identified correctly. Using all four criteria, 40 of 43 glands harboring tumor were identified, a statistically significant improvement in sensitivity (P less than 0.05). Specificity of both methods was similar (P greater than 0.1). The three tumors that were missed measured 0.5 x 0.5 x 0.5 cm, 0.5 x 0.5 x 0.5 cm, and 0.3 x 0.4 x 0.9 cm, and were found at autopsy in patients who died of causes unrelated to the prostate. The authors conclude that systematic examination of the gland with attention to multiple criteria for abnormality can improve case finding in prostatic adenocarcinoma.

Adenocarcinoma↗
PubMed1991

Urothelial hyperplasia and neoplasia. III. Detection of nitrosamine production with different bacterial genera in chronic urinary tract infections of rats.

Various agents have been implicated in inducing urothelial cancer. Although drugs, occupational and environmental carcinogens are more widely accepted as playing a major role as urothelial carcinogens, several investigations suggest that bacteria may play a role. The mechanism of how bacteria may interact with the host to augment the development of urothelial carcinoma is not well understood. Clinically, investigators have linked the development of infection, urinary stones and indwelling catheters with urothelial cancer. Other investigators have suggested that the mechanism may be related to the production of carcinogenic compounds (nitrosamines) which can be detected during urinary tract infection. In our laboratory, we showed that rats with chronic urinary tract infections produced increasing urinary levels of N,N dimethylnitrosamine over a 24 week period and that the production correlated with hyperplasia and early neoplasia of the bladder epithelium. Three bacterial genera were used and two of these (Escherichia coli and a protein sp.) showed production of increasing levels of urinary nitrosamine and correlated with infection. The purpose of this current study is to determine if other bacterial genera and strains can also produce similar increasing nitrosamine levels in the rat model of chronic urinary tract infection and thus provide evidence that a number of bacterial genera and strains can produce nitrosamines in vivo. Also, the histology of the chronically infected bladder was examined for hyperplasia and neoplasia.

Animals↗