The structure of lamin filaments in somatic cells as revealed by cryo-electron tomography.

Metazoan nuclei are equipped with nuclear lamina - a thin layer of intermediate filaments (IFs) mostly built of nuclear lamins facing the inner nuclear membrane (INM). The nuclear lamina serves as an interaction hub for INM-proteins, soluble nuclear factors and DNA. It confers structural and mechanical stability to the nucleus, transduces mechanical forces and biochemical signals across the nuclear envelope (NE) and regulates the organization of chromatin. By using cryo-electron tomography (cryo-ET), we recently provided an unprecedented view into the 3D organization of lamin filaments within the lamina meshwork in mammalian somatic cells. Through implementation of averaging procedures, we resolved the rod and globular Ig-fold domains of lamin filaments. The density maps suggested that they assemble into 3.5 nm thick filaments. Our analysis revealed interesting structural differences between nucleoplasmic and cytoplasmic intermediate filaments, raising the question of which molecular cues define their assembly modes inside the cell.

AFM imaging in solution of protein-DNA complexes formed on DNA anchored to a gold surface.

A chemical procedure for anchoring DNA molecules to gold surfaces was used to facilitate the imaging of DNA and DNA-protein complexes in buffer solution by tapping mode atomic force microscopy (TMAFM). For preparing flat gold surfaces, a novel approach was employed by evaporating small amounts of gold onto freshly cleaved mica to give flat films that were stable under aqueous buffer conditions. The thickness of the investigated films ranged from 1 to 10 nm. For typical films of 4-6 nm, which were stable under aqueous buffer conditions, the root mean square (RMS) roughness ranged between 0.25 and 0.5 nm, as measured by atomic force microscopy (AFM). This roughness is comparable to that of obtained by the template stripped gold (TSG) technique, which is widely used in scanning probe microscopy but involves more preparation steps. In order to visualize DNA and DNA-protein complexes by TMAFM, the DNA was chemisorbed to the gold surface through a linker carrying a terminal thiol group at the 5'-end of each of the DNA strands. The modified DNA fragments were bound to the gold films and imaged in buffer solution, while unmodified DNA could not be visualized. Since the DNA was not dried during the process, it can be assumed that its native conformation was retained. This mode of anchoring did not prevent interaction with proteins, as confirmed by the observation that the topology of a complex formed by adding the protein to a surface-anchored DNA was the same as that obtained by anchoring a pre-formed complex to the gold surface. We attribute this observation to the fact that the DNA is anchored to the gold surfaces only through its ends, therefore the DNA-support interaction is minimized but imaging is still possible.

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme.

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.

Gold-tagged RNA-A probe for macromolecular assemblies.

Ribonucleic acids (RNAs) play a key role in many fundamental life processes. These polymers are often found complexed with proteins in extremely large particles whose molecular mass may reach several millions of daltons (e.g., ribosomes, spliceosomes, and viruses). Structural studies of such RNA-protein complexes should help elucidate their mode of action. For the structural analyses of many macromolecular assemblies, electron microscopy (EM) has served an instrumental role. However, localization by EM of RNA within biological complexes is not yet a straightforward undertaking. Here we describe a methodology for the covalent tagging of RNA molecules with gold clusters, thereby enabling their direct visualization by microscopical methods. Our strategy involves transcription in vitro of RNAs that carry free thiol groups, using ribonucleoside triphosphate analogs containing a substituent with a terminal thiol group on their heterocyclic ring. This synthesis is followed by coupling of gold clusters to the thiolated transcript through a maleimido group. Visualization of such gold-tagged RNAs by transmission electron microscopy showed spots of gold clusters, with a diameter of 1-2 nm, arranged at nearly regular distances on an imaginary curve that presumably corresponds to the RNA chain. This assignment was corroborated by atomic force microscopy that exhibited images of RNA chains in which knob-like structures, whose height corresponds to the diameter of the gold clusters, were clearly seen. This study demonstrates the potential use of nucleic acids that are covalently labeled with gold clusters for the structural characterization of protein-RNA complexes.

Synaptotagmin II negatively regulates Ca2+-triggered exocytosis of lysosomes in mast cells.

Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcepsilonRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II-regulated secretion from lysosomes may play an important role in mast cell biology.

Basic secretagogues activate protein tyrosine phosphorylation and release of arachidonic acid in mast cells via a novel protein kinase C and phosphatidylinositol 3-kinase-dependent mechanism.

Mast cells play a central role in inflammatory and immediate-type allergic reactions. These granulated cells release by a process of regulated exocytosis a variety of biologically active substances which are either preformed (e.g. histamine), or synthesized de novo following activation [e. g. metabolites of arachidonic acid (AA) and multifunctional cytokines]. Exocytosis in mast cells is activated either in response to aggregation of the receptors for immunoglobulin E (FcepsilonRI) or by the direct activation of pertussis toxin-sensitive G-proteins by a class of receptor mimetic agents, collectively known as basic secretagogues of mast cells. In the present study we show that compound 48/80 (c48/80), a synthetic member of the class of basic secretagogues, stimulates protein tyrosine phosphorylation of a number of as yet unidentified cellular substrates. These phosphorylations were inhibited by the tyrphostin AG-18, by the phosphatidylinositol 3-kinase inhibitor wortmannin and by the protein kinase C inhibitors K252a and GF1 09203X. These inhibitors also inhibited the release of AA induced by c48/80 but had no effect on exocytosis. Taken together, our findings suggest that basic secretagogues induce protein tyrosine phosphorylation as part of their parallel multiple signaling pathways which are presumably mediated by more than one G-protein. Both protein kinase C and phosphatidylinositol 3-kinase serve as intermediates in this signaling pathway. The protein tyrosine kinase signaling pathway, which mediates the activation of AA release, does not contribute to secretion of the preformed mediators such as histamine, but it might largely contribute to the de novo production of inflammatory mediators like leukotrienes and prostaglandins.

DNA ploidy in papillary carcinoma of the thyroid gland in children and adolescents.

OBJECTIVE: To evaluate DNA ploidy in papillary thyroid carcinoma in children in correlation to the clinical course of the disease. METHODS: Flow cytometric DNA ploidy measurements were performed on formalin-fixed, paraffin-embedded tumor specimens from 14 children and 14 adult patients with papillary carcinoma of the thyroid gland. Analysis of DNA content was performed blind to patient's age and clinical presentation. RESULTS: Seven patients presented with cervical metastasis, one patient had distal metastasis and four patients had local invasion. All patients underwent total thyroidectomy. Seven children underwent bilateral modified neck dissection. Twenty-five tumors expressed diploid DNA content. No statistically significant difference in DNA content was observed between the tumors from child and adult patients. No correlation was found between DNA content and aggressive presentation in the pediatric group. CONCLUSION: Our primary results indicate that diploid DNA content is common in papillary thyroid carcinoma in children and aggressive clinical presentation is not associated with DNA aneuploidy. Larger prospective studies and long-term clinical follow-up is warranted to document the clinical significance of these observations.

Comparison of nuclear DNA content in locally invasive and noninvasive papillary carcinoma of the thyroid gland.

Extrathyroidal invasion of papillary carcinoma of the thyroid gland has a very bad prognosis. A retrospective study was performed on 40 specimens from patients with papillary carcinoma of the thyroid gland to find out whether DNA ploidy correlated with aggressive tumor behavior. The nuclear DNA content of 20 locally aggressive papillary thyroid carcinomas was studied by flow cytometry. The results were compared with those of a matched control group of 20 patients with noninvasive papillary tumors. Forty percent of the tumors with spread to extrathyroid tissue were aneuploid, whereas all the tumors without such extension were diploid. This difference was statistically significant (p < 0.003). The data suggest that the differentiation of locally noninvasive and invasive papillary thyroid carcinomas may be potentially possible by nuclear DNA determination.

Anti-neoplastic activity of paclitaxel on experimental superficial bladder cancer: in vivo and in vitro studies.

The effects of intravesical administration of paclitaxel (taxol) in a bladder tumor model in mice, as well as the drug's in vitro activity on the same tumor cells, have been studied. Two cell lines, derived from MBT-2 cells, were employed in these experiments. The T50 line (obtained by many passages in mice) was much more aggressive in vivo than the T5 line. In vivo paclitaxel treatment for 3 days after T5 implantation resulted in a considerable retardation of tumor growth, whereas under the same conditions the T50 line was much less, although still significantly, affected. When treatment was started 1 day after tumor implantation, both tumor variants were affected by paclitaxel to the same extent. The in vitro experiments utilized the MiCK assay, which allows continuous recording of the kinetics of cell growth. These studies revealed a 39.8% inhibition of cell growth by 2.10(-8)M paclitaxel in the T50 line and a 30-fold increase in concentration had only a small additional effect on the degree of inhibition. At 2.10(-8)M paclitaxel, growth of T5 was inhibited by 21.7%, which increased to 35.2% at 6.10(-7)M. The treated cells displayed bundles of microtubuli, as described for other paclitaxel-treated cells.

Automated electron tomography of large nuclear RNP (InRNP) particles--the naturally assembled complexes of precursor messenger RNA and splicing factors.

Splicing of nuclear pre-mRNA is an important step in the regulation of gene expression as only correctly spliced mRNAs will be exported to the cytoplasm to function in protein synthesis. Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA, are packaged in multicomponent large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200S region in sucrose gradients, contain all U snRNPs required for pre-mRNA splicing and several protein splicing factors, including U2AF and the SR proteins and can thus be viewed as naturally assembled complexes of pre-mRNA and splicing factors. We have previously reconstructed the three-dimensional image of negatively stained individual lnRNP particles by automated electron tomography. The reconstruction revealed a compact structure, 50 nm in diameter, composed of four major subunits. Here we further analyzed the reconstructed models and the apparent connectivity between the subunits using a new rendering technique. The uniformity of the lnRNP particles was substantiated by measurement of the volume engulfed by their surface. This study further supports the model proposed for the packaging of nuclear pre-mRNAs in lnRNP particles, where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multiintronic pre-mRNAs, and with the fact that the splicing of multiintronic pre-mRNAs does not occur in a sequential manner.

Treatment of experimental mouse bladder tumour by LPS-induced epithelial cell shedding.

The purpose of the present study was to explore the therapeutic potential of serial administration of shedding-inducing endotoxin in a mouse tumour bladder model. The studies were conducted with two variants derived from the MBT-2 tumour namely, T5 and T50, the latter being far more aggressive than the former. It was found that T5 tumours responded to intravesical lipopolysaccharides (LPS) instillation by a considerable reduction in their pace of growth (P < 0.0001) when treatment was initiated 3 days after tumour implantation, but not when started after 7 days. The T50 variant did not respond to LPS when treated 3 days after implantation, but a considerable reduction in rate of growth occurred when treatment was started after 1-2 days. Shedding induced by intravesically instilled LPS was found to retard considerably the progression rate of experimental bladder tumour.

A comparative study of the desquamation of urothelial cells during gestation and in adults mice following moderate stress or endotoxin treatment.

The present paper deals with the comparative EM study of the detachment of mouse urinary bladder epithelial cells under various physiological conditions, namely, during gestation and in adult mice following induction by endotoxin or by exposure to moderate stress. It has been shown that desquamation during gestation involves two distinct modes, shedding of single cells and formation of apoptotic bodies. Moderate stress in adult female mice induced by constant illumination for 72 or 96 hours, results in desquamation of superficial and intermediate cells. Application of LPS is followed by desquamation of single cells and whole sheets of cells. Cell detachment involves interruption of tight junctions between neighbouring cells and formation of numerous cup shaped vesicles, multivesicular bodies and vacuoles at the base of desquamating cells. LPS induces desquamation entails delivery of lysosomal enzymes extracellulary into the intercellular space. In the areas of sloughing the bladder epithelium lack permeability barrier. Desquamated cells in the bladder lumen are mostly alive. These results clearly demonstrate the existence of specific adhesion mechanisms of detachment and desquamation of uroepithelial cells.

The role of nuclear morphometry for predicting disease outcome in patients with localized renal cell carcinoma.

BACKGROUND: More than one-third of patients with localized renal cell carcinoma (RCC) will have disease progression after nephrectomy. Present histopathologic variables cannot accurately predict the outcome of individual patients. METHODS: Nuclear morphometry was performed by an image analyzer on histologic sections from 39 specimens of pathologic T1 and T2 classification RCC. All patients underwent radical nephrectomy and were followed for a mean of 7.6 years. A univariate analysis and then a multivariate stepwise regression method were used to correlate results with patients' outcome. RESULTS: The best predictors of disease free interval were mean nuclear elongation factor (MNEF) (P = 0.023), mean nuclear regularity factor (MNRF) (P = 0.034), and mean nuclear area (MNA) (N = 0.038). Univariate analysis identified a significant correlation between patient survival and MNEF (P = 0.009), MNRF (P = 0.020) and MNA (P = 0.023). Combination of MNEF and MNA was even more strongly associated with survival (P = 0.0013). Multivariate analysis revealed that MNA (P = 0.044) and MNEF (P = 0.045) correlated independently with survival. CONCLUSION: These results suggest that nuclear morphometry provides objective independent prognostic information for patients with localized RCC.

Significance of DNA ploidy in the treatment of T1 glottic carcinoma.

OBJECTIVE: To assess the role of DNA ploidy as a predictor of radioresistance in T1 glottic carcinoma. DESIGN: Case-control study. Flow cytometric DNA ploidy measurements were performed on formalin-fixed paraffin-embedded tumor specimens from 15 patients with T1 glottic laryngeal carcinomas in whom radiotherapy had failed and from a matched group of 15 patients in whom an identical radiotherapy regimen was curative. Analysis of DNA content was performed blind to outcome of treatment. SETTING: Academic tertiary referral medical center. PARTICIPANTS: Thirty patients with clinically staged T1, N0, M0 glottic carcinoma. INTERVENTION: All patients received radiation to the larynx through opposed lateral ports at a total dose of 64 to 70 Gy. RESULTS: Ten diploid and five aneuploid histograms were found in the resistant group, and six diploid and nine aneuploid histograms were found in the radiosensitive group. This difference was not statistically significant. A trend toward a higher relapse rate after radiotherapy (62.5%) among patients with diploid tumor compared with those with aneuploid tumor (35.7%) was noted. CONCLUSIONS: DNA ploidy did not predict response to radiotherapy in patients with T1 glottic cancer, probably because of the small number of patients. A trend toward lower risk of local recurrence after radiotherapy in aneuploid tumors was noted. A larger prospective study is needed to assess the value of DNA ploidy in the treatment of early laryngeal cancer.

Testicular seminoma: clinical significance of nuclear deoxyribonucleic acid ploidy pattern as studied by flow cytometry.

PURPOSE: We evaluated the clinical significance of deoxyribonucleic acid (DNA) ploidy pattern as a predictor of prognosis in patients with testicular seminoma. MATERIALS AND METHODS: Flow cytometric nuclear DNA analysis was performed on archival specimens from 65 patients with pure seminoma who underwent radical orchiectomy between 1970 and 1992. RESULTS: A total of 42 specimens (65%) exhibited a DNA diploid pattern, while 23 (35%) were DNA aneuploid. Diploidy was manifested in 73% of the stage I tumors versus 31% of stage II cancers (p = 0.004). No correlation was found between ploidy and histological type, size or local extension of the tumor. Tumor progression was observed in 5 patients, exclusively displaying aneuploid histograms (p = 0.0017), and 3 of them subsequently died of the disease. CONCLUSIONS: DNA ploidy pattern may provide important prognostic information for patients with testicular seminoma.

Moderate stress protects female mice against bacterial infection of the bladder by eliciting uroepithelial shedding.

We have previously shown (M. Aronson, O. Medalia, D. Amichay, and O. Nativ, Infect. Immun. 56:1615-1617, 1988) that shedding of viable uroepithelial cells (elicited by invading microorganisms) constitutes an antimicrobial defense mechanism. The present study deals with two different stress-involving procedures, in which increased uroepithelial shedding rendered female mice resistant to vesical infection. Moderate stress was induced in female mice by exposing the animals either to constant illumination for 96 h or to 37 degrees C heat for 24 h. In both cases, the rate of infection was considerably reduced as a result of increased epithelial shedding (P < 0.0001). Stress was manifested by both reduced thymic weight and increased blood corticosterone levels. Shedding was also elicited by intraperitoneal injection of norepinephrine together with hydrocortisone or by intravesical injection of corticosterone. Constant illumination as well as heat enormously facilitated the migration of polymorphonuclear cells into the bladder following the action of chemotactic stimuli. Male mice subjected to identical stress-generating conditions did not display considerable epithelial shedding or increased migration of polymorphonuclear cells, and they were not protected from intravesical infection.

Enhanced cytologic detection of early stage mouse bladder tumor following induction of uroepithelial cell shedding.

Previous studies from our laboratory have shown that some Escherichia coli endotoxins are capable of inducing massive normal urothelial cell shedding. In the present study we investigated whether endotoxin-induced shedding improves cytologic detection of early stage mouse bladder cancer. Mouse bladder tumor (MBT-2) cells were implanted intravesically in the submucosa of C3H female mice. Ten to 21 days later the bladders were irrigated with saline followed by instillation of endotoxin. The bladder contents of each mouse were aspirated and examined cytologically together with the bladder wash specimen. Shedding of epithelial cells was observed in only 32% of the saline irrigated specimens compared with 93% after endotoxin instillation (p < 0.00001). Analysis showed an overall accuracy rate of 39% after saline barbotage versus 78% following endotoxin administration (p < 0.00001). These results indicate that intravesical instillation of specific bacterial endotoxin significantly increases the extent of cytologic detection of early stage superficial bladder cancer.

Localized renal cell carcinoma treated by radical nephrectomy. Influence of pathologic data and the importance of DNA ploidy pattern on disease outcome.

BACKGROUND: The course of patients with renal cell carcinoma may be considerably different. Approximately 50% with presumed localized disease have metastases after nephrectomy. Pathologic stage at diagnosis, histologic grade, and histologic type have been considered the most important predictors of prognosis. Nevertheless, subsets of patients within a specified stage and grade may have considerable differences in disease progression and survival. METHODS: Flow cytometric nuclear DNA analysis was used to study pathologic Stage I or II renal cell carcinoma in 54 patients who underwent radical nephrectomy between 1974 and 1983. RESULTS: Sixty-three percent of the tumors were diploid, and 37% aneuploid. A DNA diploid pattern was more common among Stage I tumors than Stage II tumors (69% versus 33%; P < 0.04). Progression occurred in 31% of the diploid tumors, whereas among the aneuploid group the progression rate reached 59% (P < 0.06). Considered as a single indicator, DNA ploidy pattern was strongly associated with patient survival. Ten years after surgery 79% of the patients who had diploid tumors and 50% of those with aneuploid tumors were alive (P < 0.02). CONCLUSIONS: Nuclear DNA ploidy may serve as an important prognostic variable for patients with early stage renal cell carcinoma.