LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells.

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.

[A pocket pRb mutation induces the increase in its affinity to E2F4 coupled with activation of muscle differentiation].

Co-ordination of proliferation and differentiation in cells committed to muscle fate requires the interaction of the retinoblastoma gene product (pRb) with transcription factors of the E2F family. pRb has different affinities for distinct E2Fs, however, the mechanism involved in pRb-E2Fs interaction has not been completely investigated. We have found that pRb carrying a small deletion at the end of the T antigen binding region (deltaS/N), and unable to interact with large T antigen, could induce acute cell cycle block, stable prolongation of the cell cycle in G0/G1 and G2/M phases and suppression of the growth of tumor cells. The deltaS/N showed increased affinity for E2F4, bound hyperphosphorylated forms of E2F4 and induced its nuclear compartmentalization. The ability of deltaS/N to form complexes with E2F4 on DNA was associated with an increase in formation of "free" E2F4 and transsuppression of the specific reporter through preferential binding to E2F4 but not t o E2F1. Stable expression of deltaS/N in multi-potent fibroblasts promoted early muscle commitment. The results obtained suggest that a mutation in the T antigen binding region may increase in affinity of the pRb for E2F4 combined with activation of muscle differentiation.

[Drosophila melanogaster gene Merlin interacts with the clathrin adaptor protein gene lap].

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer(DBB) together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.

[Role of the porcupine gene in the development of the wing imaginal disk of Drosophila melanogaster].

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.

The correlation between pretreatment serum hormone levels and treatment outcome for patients with prostatic cancer and bony metastasis.

OBJECTIVE: To evaluate whether pretreatment serum hormone levels are a prognostic factor for prostatic cancer with bony metastasis under hormonal treatment. PATIENTS AND METHODS: Between 1980 and 1994, 96 patients with prostate cancer and bony metastasis were included for an evaluation by a retrospective review of their charts. All 96 had received hormonal treatment after a diagnosis of metastatic prostatic carcinoma. Serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were assessed before treatment. The patients were divided into two groups according to their response during the follow-up. Group 1 (good response) had no change or resolution of metastatic lesion(s) on the bone scan and a declining prostate-specific antigen (PSA) level. Group 2 had increased PSA or progression of metastatic lesion(s) on the bone scan. Tumours were graded as low (2-4), intermediate (5-7) and high (8-10) using the Gleason score. RESULTS: There were 43 patients in group 1 and 53 in group 2; the overall mean (sd) age was 72.5 (6.8) years and the follow-up 29.5 (0.5) months. The respective mean (sd) levels of testosterone, LH, FSH and prolactin before treatment were 4.6 (1.6) ng/mL, 20.2 (13.3) mIU/mL, 19.6 (18.6) mIU/mL and 20.7 (12.1) ng/mL in group 1, and 2.6 (1.0) ng/mL, 27.3 (11.0) mIU/mL, 27.1 (9.8) mIU/mL and 41.3 (28.4) ng/mL in group 2. The level of testosterone was significantly higher in group 1 than in group 2, while LH, FSH and prolactin were significantly lower in group 1 than in group 2. When stratified by tumour grade, patients in group 1 still had significantly higher pretreatment testosterone and lower LH, FSH and prolactin than those in group 2. CONCLUSION: Higher testosterone and lower LH, FSH and prolactin levels were good prognostic factors for patients with metastatic prostatic cancer under hormonal treatment, irrespective of tumour grading.

Expression of A chain and B chain of beta-bungarotoxin from taiwan banded krait: the functional implication of the interchain disulfide bond between A chain and B chain.

beta-Bungarotoxin (beta-Bgt), the main presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains, the A chain and B chain, cross-linked by an interchain disulfide bond. The A and B chain cDNAs were subcloned into expression vectors pT7-7 and pET20b(+), respectively, and transformed into Escherichia coli strain BL21(DE3). The expressed protein was isolated from the inclusion bodies of E. coli and subjected to refolding into its folded structure. The yields of the refolded A and B chains increased markedly by at least 100-fold after substituting Ser for Cys1S of A chain and Cys55 of B chain, which formed an interchain disulfide bond. Either the A(C15) chain or B(C55S) chain alone or in combination cannot exhibit the phospholipase A2 activity or synaptosome binding activity of beta-Bgt. Nevertheless, the results of competitive enzyme-linked immunoassay, CD spectra, and fluorescence measurement revealed that the A(C15S) chain and B(C55S) chain possessed a native-like structure like the subunits of native beta-Bgt. Moreover, the interfacial interaction between the A and B chains explored by glutaraldehyde cross-linking revealed the essential aspects of the intact interchain disulfide bond in this interaction. This suggests that the formation of the interchain disulfide bond should not be a crucial step for the formation of folded A and B chains in the venom glands, and that the integrity of the interchain disulfide linkage favors the subunit interaction that consequently fulfills the functional mechanism of beta-Bgt.

High failure rate using allograft fascia lata in pubovaginal sling surgery for female stress urinary incontinence.

OBJECTIVES: To present our unfavorable experiences using allograft fascia lata. Allograft fascia lata is an attractive sling material providing less pain, a shorter operation time, and a reported effectiveness equal to autologous fascia. METHODS: A total of 18 women (mean age 51.7 years, range 37 to 76) underwent pubovaginal sling surgery for stress urinary incontinence between March 1999 and July 1999 and were enrolled in this study. Solvent dehydrated gamma-irradiated human fascia lata with a size of 7 x 2 cm was used as the sling. The results were collected with a questionnaire survey. RESULTS: All patients were followed up for a mean of 9.2 months (range 6.9 to 11.6). Thirteen patients considered the surgery successful or to have provided improvement, with a mean of 82.5% (range 50% to 100%) subjective improvement. Five patients (27.8%) had significant failure with full recurrence of incontinence within 3 to 6 months. CONCLUSIONS: Solvent dehydrated gamma-irradiated allograft fascia is not reliable in pubovaginal sling surgery. The high failure rates within a short period prohibit its use in the operative management of stress urinary incontinence.

Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction.

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.

Effects of hyperprolactinemia on testosterone production in rat Leydig cells.

The pathogenesis of hyperprolactinemia (hyperPRL) induced hypogonadism has been suggested to be related with a dysfunction of hypothalamus-pituitary-testis axis. While the direct inhibitory effects of prolactin (PRL) on testosterone (T) release have been demonstrated, the mechanism is still unclear. Our previous study demonstrated a diminished T release in the testicular interstitial cells (TICs) from the anterior pituitary (AP)-grafted rats as compared with the control, and the pattern was in agreement with the in vivo model. However, TICs incubation cannot totally represent the response of the Leydig cells. Therefore, a Percoll gradient purified Leydig cell model was adopted to explore the response of T release under similar challenges in this study to investigate the effects of hyperPRL on the Leydig cells per se. HyperPRL in male rats was induced by grafting rat AP under the renal capsule. The control animals were grafted with rat brain cortex tissue (CX). Six weeks after grafting, the rats were sacrificed. Either TICs or Leydig cells were isolated, respectively, for in vitro incubation and challenge. Challenge drugs included human chorionic gonadotropin (hCG, 0.05 IU/ml), steroidogenic precursors (25-OH-cholesterol, 10(-6) M; pregnenolone, 10(-6) M), forskolin (an anenylyl cyclase activator, 10(-4) M) and 8-bromo-3':5' cyclic adenosine monophosphate (cAMP) (8-Br-cAMP 10(-4) M). T released by TICs or Leydig cells was determined by radioimmunoassay. The TICs from the AP-grafted rats showed lower levels of T release than the control group while the purified Leydig cells demonstrated a reverse pattern in response to challenges of hCG, steroidogenic precursors, forskolin and 8-Br-cAMP. In hyperPRL rats, a paradoxical pattern of T release between TICs and purified Leydig cells is observed. The purified Leydig cells from AP-grafted rats demonstrated a higher level amount of T release than the control after stimulation. The phenomenon can be attributed to the change of Leydig cell sensitivity to the stimulation after the effects of chronic hyperPRL. Moreover, another possibility is the role played by other interstitial cells to modulate steroidogenesis in Leydig cells.

Mannitol facilitates rabbit urinary bladder recovery from overdistension injury.

OBJECTIVES: To investigate the existence and functional significance of the enhanced lipid peroxidation in bladder overdistension injury and to explore the effect of mannitol, a free radical scavenger. METHODS: Overdistension of rabbit bladders was induced and maintained for 3 hours by infusing normal saline into the bladder while keeping the intravesical pressure at 30 cm H(2)O. The bladders were then emptied and decompressed. Intravenous 20% mannitol was initiated 5 minutes before decompressing the overdistension. Detrusor tissue was obtained from the following groups: control, at the end of the overdistension period, and 30 minutes, 2 hours, and 7 days after decompressing the bladder. The tissue level of adenosine triphosphate (ATP) and phosphocreatine (PCr) and the lipid peroxidation product malondialdehyde (MDA) was assayed. Detrusor contractility was assessed by the response of the detrusor strips to KCl and bethanechol. RESULTS: Decompressing the overdistended bladder led to a period of enhanced lipid peroxidation with an increase of MDA content from 225 to 384 pmol/mg protein 30 minutes after the decompression. Two hours later, the MDA content had recovered to the normal level. Mannitol abolished this period of enhanced lipid peroxidation. Overdistension impaired detrusor contractility and reduced the content of PCr (from 24.1 to 10.8 nmol/mg protein) and ATP (from 9.6 to 4.6 nmol/mg protein). Both detrusor contractility and the content of PCr and ATP further decreased 30 minutes after the decompression (PCr 5.4 nmol/mg, ATP 2.8 nmol/mg). They had recovered, but not fully, 7 days later. Mannitol prevented the further decrease in detrusor contractility and in the content of PCr and ATP during the initial decompression period (30 minutes after the decompression). In addition, the mannitol-treated group had quicker recovery in PCr and ATP levels, which returned to normal 7 days later. CONCLUSIONS: Decompressing an overdistended bladder leads to enhanced lipid peroxidation, which is associated with an additionally decreased energetic metabolism and a more impaired contractile function. Mannitol effectively prevents enhanced lipid peroxidation and facilitates functional recovery. These results show that reactive oxygen species play a significant role in bladder overdistension injury.

Analysis of the human neurofibromatosis type 2 gene promoter and its expression.

OBJECTIVE: It is hypothesized that transcriptional regulation plays an important role for neurofibromatosis type 2 (NF2) expression in Schwann cells and other cell types. The objective of this study is the isolation and characterization of the transcriptional regulatory elements of the NF2 gene. STUDY DESIGN AND SETTING: A bacterial artificial chromosome library and a partial genomic DNA library were used to isolate the human NF2 gene; NF2 promoter-luciferase constructs were generated, and promoter activities were assayed. This study was carried out in a molecular biology laboratory. RESULTS: A bacterial artificial chromosome clone with an approximately 100-kilobase insert containing nearly the entire human NF2 gene has been isolated. An additional 5' NF2 sequence has also been cloned. Transient transfection experiments demonstrate strong promoter activity from the NF2 5' flanking DNA. CONCLUSIONS: The NF2 gene is approximately 100 kilobases long. Both positive and negative regulatory elements are present in NF2 5' flanking regions. SIGNIFICANCE: Better understanding of the NF2 gene and its regulation will improve molecular diagnostics and ultimately treatment of patients with NF2.

Effects of aging on mitochondrial enzyme activity of rat urinary bladder.

OBJECTIVES: Our previous study showed that aged rat bladders became fatigued faster than young bladders following repeated contraction induced by electrostimulation. One factor might be a lower energy-producing capability secondary to a decreased mitochondrial enzyme activity of the aged bladder. This study examined this possibility. MATERIALS AND METHODS: Mitochondria from 3- (n = 11) and 24-month-old (n = 10) Sprague-Dawley rats were isolated. Activities of the following enzymes were assayed: two key enzymes in the citric acid cycle, citrate synthase and malate dehydrogenase, and three enzymes in the respiratory chain reaction, NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome c oxidase. The concentration of phosphocreatine and ATP in the aged rat bladders and a separate group of young bladders (n = 12) was determined using high-performance liquid chromatography. RESULTS: (1) The aged bladders have a significantly lower level of phosphocreatine and ATP content than those of young bladders. (2) The activities of all five enzymes assayed were significantly lower in the aged bladders than in young bladders, especially for citrate synthase, which had only 46.8% of the activity of young bladders. CONCLUSIONS: Aging reduces the mitochondrial enzyme activity of the rat bladder resulting in a lower energy-production capability, which might explain some of the voiding dysfunctions found in the elderly.

p53 dependence of topoisomerase I recruitment in vivo.

DNA damage is attended by rapid recruitment of endogenous type I topoisomerase (topo I) into covalent cleavage complexes with genomic DNA in vivo. In contrast, endogenous topoisomerase II alpha and beta are not stimulated by DNA damage. We show that topo I and p53 are able to associate at arrested topo I-genomic DNA covalent complexes in vivo, suggesting that p53 directly stimulates topo I activity and damage to the genome of the afflicted cell. Moreover, cells that express wild-type p53 are most proficient at recruiting topo I after DNA damage; however, the p53 dependence is conditional because topo I recruitment after DNA damage can be restored if p53 mutant cells (containing a single mutant allele) are artificially held in G1. In contrast, p53 null mutants do not recruit topo I after DNA damage under any conditions (although camptothecin-dependent topo I/DNA complexes readily form in the nulls). These results show that topo I activation after DNA damage depends on the p53 status of the cell. It also depends upon the cell cycle in a way that is very different from that observed with DNA replication-dependent, camptothecin-mediated DNA breaks. The data suggest a model where p53 activates topo I, which inflicts additional genomic damage after the initial UV damage events. Topoisomerases therefore contribute to the p53 commitment to apoptosis, and topo I might assist in elimination of DNA-damaged cells as part of the cellular proofreading function inherent in the p53 pathway.

Disruption of the actin cytoskeleton leads to inhibition of mitogen-induced cyclin E expression, Cdk2 phosphorylation, and nuclear accumulation of the retinoblastoma protein-related p107 protein.

The actin cytoskeleton has been found to be required for mitogen-stimulated cells to passage through the cell cycle checkpoint. Here we show that selective disruption of the actin cytoskeleton by dihydrocytochalasin B (H(2)CB) blocked the mitogenic effect in normal Swiss 3T3 cells, leading to cell cycle arrest at mid to late G(1) phase. Cells treated with H(2)CB remain tightly attached to the substratum and respond to mitogen-induced MAP kinase activation. Upon cytoskeleton disruption, however, growth factors fail to induce hyperphosphorylation of the retinoblastoma protein (pRb) and the pRb-related p107. While cyclin D1 induction and cdk4-associated kinase activity are not affected, induction of cyclin E expression and activation of cyclin E-cdk2 complexes are greatly inhibited in growth-stimulated cells treated with H(2)CB. The inhibition of cyclin E expression appears to be mediated at least in part at the RNA level and the inhibition of cdk2 kinase activity is also attributed to the decrease in cdk2 phosphorylation and proper subcellular localization. The expression patterns of cdk inhibitors p21 and p27 are similar in both untreated and H(2)CB-treated cells upon serum stimulation. In addition, the changes in subcellular localization of pRb and p107 appear to be linked to their phosphorylation states and disruption of normal actin structure affects nuclear migration of p107 during G(1)-to-S progression. Taken together, our results suggest that the actin cytoskeleton-dependent G(1) arrest is linked to the cyclin-cdk pathway. We hypothesize that normal actin structure may be important for proper localization of certain G(1) regulators, consequently modulating specific cyclin and kinase expression.

Genetic organization of A chain and B chain of beta-bungarotoxin from Taiwan banded krait (Bungarus multicinctus). A chain genes and B chain genes do not share a common origin.

beta-Bungarotoxin, the main presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains, the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, A and B chain genes isolated from the liver of B. multicinctus encoded the A and B chain precursors, respectively. Analyses of the coding regions of the A and B chain genes revealed that both consist of three exons and two introns. The sequences of all exon/intron junctions agree with the GT/AG rule. However, sequence alignment and phylogenetic analysis did not support that the evolution of A and B chain genes are closely related. Comparative analysis of A chain genes with Viperinae and Crotalinae phospholipase A2 genes indicated that genetic divergence of the A chain and phospholipase A2s was in accordance with their family. Moreover, evolutionary divergence of the intron and exon regions of the A chain, as observed for phospholipase A2 genes, was not consistent. Noticeably, the transcription of A and B chain genes may be regulated under different transcription factors as revealed by analyses of their promoter sequences. In terms of the finding that A and B chains are encoded separately by different genes, this strongly supports the view that the intact beta-bungarotoxin molecules should be derived from the pairing of A and B chains after their mRNAs are translated.

Assessment of a three-dimensional operating system with skill tests in a pelvic trainer.

OBJECTIVES: To compare the performance of laparoscopic skill assisted by a traditional two-dimensional (2D) and a three-dimensional (3D) endoscopic video system in a pelvic trainer. MATERIALS AND METHODS: The 3D imaging system (DeepVision((R)), Automated Medical Products Corp.) consists of a traditional single lens optic laparoscope, a light source, an endoscopic camera (Stryker), a DeepVision processor and a DeepVision monitor. The 2D images could be obtained with the same system without turning on the DeepVision processor. Thirty-four medical personnel with no laparoscopic surgical experience were enrolled to perform two skill tests, the object-pick-up and spatial orientation test in a trainer box. They were randomly divided into two groups, one group performed the test under 2D conditions first and 3D later, and another group performed the test under 3D conditions first and 2D later. The duration needed to complete the skill tests was recorded and the differences on performance time under 2D and 3D conditions were calculated for each participant. Two-way ANOVA was used to analyze the statistic difference on the performance time in two conditions. RESULTS: The duration needed to complete the initial skill tests was similar among 2D and 3D conditions. For both tests, the average performance time decreased significantly for the second attempt regardless of 2D or 3D conditions. Statistic analysis disclosed significant difference for learning factor (p < 0.001 for object-pick-up test and p < 0.01 for spatial orientation test), but no significant difference between 2D and 3D conditions (p = 0.276 for object-pick-up test and p = 0.327 for spatial orientation test). CONCLUSION: A significant decrease of the performance time at the second attempt reflected the importance of a learning process in laparoscopic surgery. It appears that no significant benefits were obtained by this 3D operating system for surgeons without laparoscopic surgical experience.

Mitochondrial DNA deletion of the human detrusor after partial bladder outlet obstruction-correlation with urodynamic analysis.

OBJECTIVES: To investigate mitochondrial DNA (mtDNA) mutations in human detrusor after partial bladder outlet obstruction (BOO) and correlate the findings with the results of urodynamic studies. METHODS: Sixty-two male patients with and without BOO were recruited and assessed by the International Prostate Symptom Score, a quality-of-life assessment index, and sonography. The severity of partial BOO was determined by pressure-flow study with an International Continence Society (ICS) nomogram. Random detrusor biopsies obtained cystoscopically were analyzed by polymerase chain reaction (PCR) techniques to detect possible mtDNA deletions. Primer-shift PCR and DNA sequencing were then performed to characterize specific mtDNA deletions. A semiquantitative PCR method was used to determine the proportion of the deleted mtDNA in detrusor. Finally, the mtDNA deletion and the urodynamic results were compared statistically. RESULTS: A 4977-bp mtDNA deletion was identified in the human detrusor. Its incidence and proportion were found to increase after partial BOO (P = 0.005 and 0.012, respectively). The incidence of the mtDNA deletion was 4.2% (1 of 24) in the unobstructed group, 27.8% (5 of 18) in the equivocal group, and 40% (8 of 20) in the obstructed group. The mean proportion of the 4977-bp deleted mtDNA was 23.7 and 12.7 times higher in the obstructed and equivocal groups, respectively, compared with that of the unobstructed group. CONCLUSIONS: We found mtDNA with the 4977-bp deletion in human detrusor and an increase of this deletion after partial BOO. This molecular change might account for the previous observations of mitochondrial functional impairment and voiding dysfunction after partial BOO.

The correlation between clinical outcome and residual prostatic weight ratio after transurethral resection of the prostate for benign prostatic hyperplasia.

OBJECTIVE: To assess in a prospective study the use of a new variable, the residual prostatic weight ratio (RPWR), for evaluating the clinical outcome after transurethral resection of the prostate (TURP). PATIENTS AND METHODS: From April 1996 to June 1997, 40 men (mean age 70.4 years, range 53-85) with symptomatic benign prostatic hyperplasia were evaluated using the American Urological Association (AUA) symptom score, measurements of the mean and maximum urinary flow rate (Qave and Qmax), and by transrectal ultrasonography (TRUS) before and 16 weeks after TURP. The estimated total prostate weight was derived as 0.52 x length x width x height x the specific gravity of the prostate (1.010). The RPWR was calculated as the prostate weight after TURP divided by the initial weight, where the value after TURP was the initial weight minus that of the TURP specimen. The clinical outcome was evaluated by the difference (Delta) in AUA score, Qmax and Qave before and 16 weeks after surgery. RESULTS: There was a close correlation between the estimated prostate weight and the actual weight of the TURP specimen (r = 0.82 and 0.80 for the adenoma and total prostate, respectively). The mean (SD) RPWR, DeltaAUA score, DeltaQmax and DeltaQave were 50.1 (17.1)%, 11.5 (5. 3), 9.0 (4.2) mL/s and 6.2 (2.9) mL/s, respectively. There was a negative correlation between the RPWR and the DeltaAUA, DeltaQmax and DeltaQave (r = -0.81, -0.68, and -0.70, respectively, P < 0.05). The prostate volume estimated by TRUS decreased significantly 16 weeks after TURP. CONCLUSIONS: TRUS is a useful tool for estimating prostate weight before surgery. The smaller the RPWR at 16 weeks after TURP, the better the clinical outcome.