OBJECTIVE

To evaluate our long-term experience with patients treated uniformly with radical cystectomy and pelvic lymph node dissection for invasive bladder cancer and to describe the association of the primary bladder tumor stage and regional lymph node status with clinical outcomes.

METHODS

All patients undergoing radical cystectomy with bilateral pelvic iliac lymphadenectomy, with the intent to cure, for transitional-cell carcinoma of the bladder between July 1971 and December 1997, with or without adjuvant radiation or chemotherapy, were evaluated. The clinical course, pathologic characteristics, and long-term clinical outcomes were evaluated in this group of patients.

RESULTS

A total of 1,054 patients (843 men [80%] and 211 women) with a median age of 66 years (range, 22 to 93 years) were uniformly treated. Median follow-up was 10.2 years (range, 0 to 28 years). There were 27 (2.5%) perioperative deaths, with a total of 292 (28%) early complications. Overall recurrence-free survival at 5 and 10 years for the entire cohort was 68% and 66%, respectively. The 5- and 10-year recurrence-free survival for patients with organ-confined, lymph node-negative tumors was 92% and 86% for P0 disease, 91% and 89% for Pis, 79% and 74% for Pa, and 83% and 78% for P1 tumors, respectively. Patients with muscle invasive (P2 and P3a), lymph node-negative tumors had 89% and 87% and 78% and 76% 5- and 10-year recurrence-free survival, respectively. Patients with nonorgan-confined (P3b, P4), lymph node-negative tumors demonstrated a significantly higher probability of recurrence compared with those with organ-confined bladder cancers (P <.001). The 5- and 10-year recurrence-free survival for P3b tumors was 62% and 61%, and for P4 tumors was 50% and 45%, respectively. A total of 246 patients (24%) had lymph node tumor involvement. The 5- and 10-year recurrence-free survival for these patients was 35%, and 34%, respectively, which was significantly lower than for patients without lymph node involvement (P <.001). Patients could also be stratified by the number of lymph nodes involved and by the extent of the primary bladder tumor (p stage). Patients with fewer than five positive lymph nodes, and whose p stage was organ-confined had significantly improved survival rates. Bladder cancer recurred in 311 patients (30%). The median time to recurrence among those patients in whom the cancer recurred was 12 months (range, 0.04 to 11.1 years). In 234 patients (22%) there was a distant recurrence, and in 77 patients (7%) there was a local (pelvic) recurrence.

CONCLUSIONS

These data from a large group of patients support the aggressive surgical management of invasive bladder cancer. Excellent long-term survival can be achieved with a low incidence of pelvic recurrence.

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

We have determined the structure of bovine rhodopsin at 2.65 A resolution using untwinned native crystals in the space group P3(1), by molecular replacement from the 2.8 A model (1F88) solved in space group P4(1). The new structure reveals mechanistically important details unresolved previously, which are considered in the membrane context by docking the structure into a cryo-electron microscopy map of 2D crystals. Kinks in the transmembrane helices facilitate inter-helical polar interactions. Ordered water molecules extend the hydrogen bonding networks, linking Trp265 in the retinal binding pocket to the NPxxY motif near the cytoplasmic boundary, and the Glu113 counterion for the protonated Schiff base to the extracellular surface. Glu113 forms a complex with a water molecule hydrogen bonded between its main chain and side-chain oxygen atoms. This can be expected to stabilise the salt-bridge with the protonated Schiff base linking the 11-cis-retinal to Lys296. The cytoplasmic ends of helices H5 and H6 have been extended by one turn. The G-protein interaction sites mapped to the cytoplasmic ends of H5 and H6 and a spiral extension of H5 are elevated above the bilayer. There is a surface cavity next to the conserved Glu134-Arg135 ion pair. The cytoplasmic loops have the highest temperature factors in the structure, indicative of their flexibility when not interacting with G protein or regulatory proteins. An ordered detergent molecule is seen wrapped around the kink in H6, stabilising the structure around the potential hinge in H6. These findings provide further explanation for the stability of the dark state structure. They support a mechanism for the activation, initiated by photo-isomerisation of the chromophore to its all-trans form, that involves pivoting movements of kinked helices, which, while maintaining hydrophobic contacts in the membrane interior, can be coupled to amplified translation of the helix ends near the membrane surfaces.

Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and microsequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M) downward arrow motif, a site that is representative of its enzymic specificity. In vitro peptide processing studies andor Ala substitutions of the P1-P5 sites suggested that hydrophobicaliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines. In situ hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons.

A 200-300 kb region of chromosome 3p14.2, including the fragile site locus FRA3B, is homozygously deleted in multiple tumor-derived cell lines. Exon amplification from cosmids covering this deleted region allowed identification of the human FHIT gene, a member of ther histidine triad gene family, which encodes a protein with 69% similarity to an S. pombe enzyme, diadenosine 5', 5''' P1, P4-tetraphosphate asymmetrical hydrolase. The FHIT locus is composed of ten exons distributed over at least 500 kb, with three 5' untranslated exons centromeric to the renal carcinoma-associated 3p14.2 breakpoint, the remaining exons telomeric to this translocation breakpoint, and exon 5 within the homozygously deleted fragile region. Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas.

1: The pharmacology of the stable cell line expressing human alpha(4)beta(3)delta GABA(A) receptor was investigated using whole-cell patch-clamp techniques. 2: alpha(4)beta(3)delta receptors exhibited increased sensitivity to GABA when compared to alpha(4)beta(3)gamma(2) receptors, with EC(50)'s of 0.50 (0.46, 0.53) microM and 2.6 (2.5, 2.6) microM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher efficacy at alpha(4)beta(3)delta receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. 3: The delta subunit conferred slow desensitization to GABA, with rate constants of 4.8+/-0.5 s for alpha(4)beta(3)delta and 2.5+/-0.2 s for alpha(4)beta(3)gamma(2). However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. 4: alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2) demonstrated equal sensitivity to inhibition by the cation zinc (2-3 microM IC(50)). However, alpha(4)beta(3)delta receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC(50) for GABA antagonists SR-95531 and picrotoxin, was similar for alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2). Likewise, inhibition was observed on both subtypes at high and low pH. 5: alpha(4)beta(3)delta receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on alpha(4)beta(3)gamma(2) cells, and the inverse agonist DMCM showed allosteric inhibition of alpha(4)beta(3)gamma(2) receptors. 6: The efficacy of neurosteroids at alpha(4)beta(3)delta receptors was greatly enhanced over that observed at alpha(4)beta(3)gamma(2) receptors. The greatest effect was observed using THDOC with 524+/-71.6% potentiation at alpha(4)beta(3)delta and 297.9+/-49.7% at alpha(4)beta(3)gamma(2) receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at alpha(4)beta(3)delta receptors versus alpha(4)beta(3)gamma(2) receptors. 7: We show that the alpha(4)beta(3)delta receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. British Journal of Pharmacology (2002) 136, 965-974

1. To investigate the origin and functional significance of a recently described tonic GABAA receptor-mediated conductance in cerebellar granule cells we have made recordings from cells in cerebellar slices from rats of different ages (postnatal days P4 to P28). 2. During development there was a dramatic change in the properties of GABA-mediated synaptic transmission. The contribution to GABAA receptor-mediated charge transfer from the tonic conductance (GGABA), relative to that resulting from discrete spontaneous postsynaptic currents (sPSCs), was increased from 5% at P7 to 99% at P21. GGABA was reduced by bicuculline, tetrodotoxin and by lowering extracellular Ca2+, and was initially present only in those cells which exhibited sPSCs. 3. At P7 sPSCs were depolarizing, occasionally triggering a single action potential. By P18 the GABA reversal potential was shifted close to the resting potential and GGABA produced a shunting inhibition. Removal of GGABA by bicuculline increased granule cell excitability in response to current injection. 4. This novel tonic inhibition is present despite the low number of Golgi cell synapses on individual granule cells and appears to result from 'overspill' of synaptically released GABA leading to activation of synaptic and extrasynaptic GABAA receptors.

To determine the role of dendritic filopodia in the genesis of excitatory synaptic contacts and dendritic spines in hippocampal area CA1, serial section electron microscopy and three-dimensional analysis of 16 volumes of neuropil from nine male rat pups, aged postnatal day 1 (P1) through P12, were performed. The analysis revealed that numerous dendritic filopodia formed asymmetric synaptic contacts with axons and with filopodia extending from axons, especially during the first postnatal week. At P1, 22 +/- 5.5% of synapses occurred on dendritic filopodia, with 19 +/- 5.9% on filopodia at P4, 20 +/- 8.0% at P6, decreasing to 7.2 +/- 4.7% at P12 (p < 0.02). Synapses were found at the base and along the entire length of filopodia, with many filopodia exhibiting multiple synaptic contacts. In all, 162 completely traceable dendritic filopodia received 255 asymmetric synaptic contacts. These synapses were found at all parts of filopodia with equal frequency, usually occurring on fusiform swellings of the diameter. Most synaptic contacts (53 +/- 11%) occurred directly on dendritic shafts during the first postnatal week. A smaller but still substantial portion (32 +/- 12%) of synapses were on shafts at P12 (p < 0.036). There was a highly significant (p < 0.0002) increase in the proportion of dendritic spine synapses with age, rising from just 4.9 +/- 4.3% at P1 to 37 +/- 14% at P12. The concurrence of primarily shaft and filopodial synapses in the first postnatal week suggests that filopodia recruit shaft synapses that later give rise to spines through a process of outgrowth.

Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA.

A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves. A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site-specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining.

Extinction of conditioned fear is an important model both of inhibitory learning and of behavior therapy for human anxiety disorders. Like other forms of learning, extinction learning is long-lasting and depends on regulated gene expression. Epigenetic mechanisms make an important contribution to persistent changes in gene expression; therefore, in these studies, we have investigated whether epigenetic regulation of gene expression contributes to fear extinction. Since brain-derived neurotrophic factor (BDNF) is crucial for synaptic plasticity and for the maintenance of long-term memory, we examined histone modifications around two BDNF gene promoters after extinction of cued fear, as potential targets of learning-induced epigenetic regulation of gene expression. Valproic acid (VPA), used for some time as an anticonvulsant and a mood stabilizer, modulates the expression of BDNF, and is a histone deacetylase (HDAC) inhibitor. Here, we report that extinction of conditioned fear is accompanied by a significant increase in histone H4 acetylation around the BDNF P4 gene promoter and increases in BDNF exon I and IV mRNA expression in prefrontal cortex, that VPA enhances long-term memory for extinction because of its HDAC inhibitor effects, and that VPA potentiates the effect of weak extinction training on histone H4 acetylation around both the BDNF P1 and P4 gene promoters and on BDNF exon IV mRNA expression. These results suggest a relationship between histone H4 modification, epigenetic regulation of BDNF gene expression, and long-term memory for extinction of conditioned fear. In addition, they suggest that HDAC inhibitors may become a useful pharmacological adjunct to psychotherapy for human anxiety disorders.

Electron microscopic analysis of in vitro transcriptional complexes of pBR322 and pACYC184 revealed five and six major transcriptional units, respectively, in these two plasmid vectors. These units are transcribed with various efficiencies, depending upon the individual promoter strengths, which differ in pBR322 up to 10-fold. A most interesting signal arrangement was found at the beginning of the tetracycline resistance region, where two partially overlapping promoters (P1 and P2) initiate transcription crosswise in opposite directions. Whereas P2 is known to promote tetracycline resistance and to be inactivated by HindIII cleavage, P1 is able to transcribe DNA integrated at that site and probably contributes to the expression of the beta-lactamase gene in pBR322. In pACYC184, besides P1, P2, and the cat (chloramphenicol resistance) promoter (P5), two initiation sites (P3 and P4) were mapped in a region that appears to be part of insertion sequence 1. The maps of transcription signals permit a more predictable utilization of these cloning vehicles and also allow the reinterpretation of earlier cloning results.

To understand the effect of human MHC class II polymorphism on antigen recognition, we analyzed the memory T cell response to three tetanus toxin epitopes defined by three short synthetic peptides (p2, p4 and p30). We found that p2 and p30 are universally immunogenic, since they are recognized by all primed donors, irrespective of their MHC haplotypes. The analysis of specific clones indicates that both peptides are very promiscuous in their capacity to bind to class II. p30 can be recognized in association with DRw11(5), 7, 9 and with DPw2 and DPw4, while p2 can be recognized in association with DR1, DRw15(2), DRw18 (3), DR4Dw4, DRw11(5), DRw13(w6), DR7, DRw8, DR9, DRw52a and DRw52b. On the contrary, the third peptide, p4, can be recognized by only half of the donors in association with only DRw52a and DRw52c. Analysis of truncated peptides shows that p30 contains three distinct epitopes, each recognized in association with different class II molecules. Therefore, the restriction specificity is already set at the level of the peptide-MHC complex and, in all cases, T cells discriminate p30 bound to different class II molecules. On the contrary, p2 contains only one epitope, which is recognized in association with all DR molecules. In this case we found two different restriction patterns. Some clones are monogamous, since they recognize the peptide in association with one DR allele, while others are promiscuous, since they recognize by peptide in association with several different DR molecules. Thus, in this case, the restriction specificity is also set at the level of the T cell receptor. We suggest that both the promiscuous binding of peptides and the promiscuous recognition by T cells are dependent on the particular structure of the DR molecules, having a monomorphic alpha chain associated with a polymorphic beta chain.

Recent advances in our understanding of the role of inositides in cell signalling have led to the central hypothesis that a receptor-stimulated phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) results in the formation of two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The existence of another pathway of inositide metabolism was first suggested by the discovery that a novel inositol trisphosphate, Ins(1,3,4)P3, is formed in stimulated tissues; the metabolic kinetics of Ins(1,3,4)P3 are entirely different from those of Ins(1,4,5)P3 (refs 6, 7). The probable route of formation of Ins(1,3,4)P3 was recently shown to be via a 5-dephosphorylation of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a compound which is rapidly formed on muscarinic stimulation of brain slices, and which can be readily converted to Ins(1,3,4)P3 by a 5-phosphatase in red blood cell membranes. However, the source of Ins(1,3,4,5)P4 is unclear, and an attempt to detect a possible parent lipid, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), was unsuccessful. The recent discovery that the higher phosphorylated forms of inositol (InsP5 and InsP6) also exist in animal cells suggested that inositol phosphate kinases might not be confined to plant and avian tissues, and here we show that a variety of animal tissues contain an active and specific Ins(1,4,5)P3 3-kinase. We therefore suggest that an inositol tris/tetrakisphosphate pathway exists as an alternative route to the dephosphorylation of Ins(1,4,5)P3. The function of this novel pathway is unknown.