Intranuclear microtubules are hallmarks of an unusual form of cell death in cisplatin-treated C6 glioma cells.

We describe an unusual form of non-accidental cell death marked by ectopic microtubules in the nucleus of a subpopulation of cisplatin-treated C6 glioma astrocytes in culture. At electron microscopy, the perinuclear condensed chromatin did not completely adhere to the nuclear envelope of these cells being separated by single or loosely bundled 20-nm-thick microtubules located in an electron-lucid slit-like zone; the presence of alpha-tubulin lining the inner membrane of the nuclear envelope was confirmed by immunolabeling at confocal microscopy. Since tufts of microfilaments-like fibers also occurred in their central nuclear areas, these cells are referred to as CIMMs (Cells with Intranuclear Microtubules and Microfilaments). The nuclear reorganization of CIMMs also involved nucleolar segregation and formation of heterogeneous ectopic ribonucleoprotein (RNP)-derived structures, indicating disruption of the RNP-based transcription machinery. The cytoplasmic organelles of CIMMs were structurally intact, and propidium iodide did not accumulate intracellularly under vital conditions while the plasma membrane was often Annexin V-positive. All these findings suggest that CIMMs were lethally damaged and committed to an atypical programmed cell death resembling early apoptosis (this is also supported by the presence of a limited number of TUNEL-positive CIMMs). CIMMs appeared well before the main cisplatin-induced cycling arrest of the cell population (G2/M block at 72 h) and had mostly G1 DNA content: this suggests that they may represent the cohort of cells which passed cisplatin-altered mitoses with intranuclear retention of microtubules from an incompletely disassembled mitotic spindle.

Subcellular targets of mercaptoborate (BSH), a carrier of 10B for neutron capture therapy (BNCT) of brain tumors.

The transformed C6 glial cells in cultures were treated with sodium mercaptoborate (Na(2)B(12)H(11)SH, BSH), a carrier of atomic targets ((10)B) of thermal neutrons for the neutron capture therapy of brain tumors. As shown by light microscopy, the therapeutic dose of BSH (100 microg/ml) did not alter the gross morphology and growth of the population of cells within a 72 h treatment interval. Electron microscopic analysis of these cells revealed activation of nucleoli and, occasionally, enlarged and bifurcated mitochondria. After 200 microg BSH/ml and 72 h treatment, growth of the cell population was inhibited and ultrastructural changes became more profound. They included condensation of chromatin and its allocation to the nuclear envelope which formed deeper invaginations. Mitochondria further increased in size and were characterized by slim or angular cristae. Moreover, in circumscribed segments of some of the slightly swollen mitochondria their cristae disappeared or were reduced to fine pouch-like structures localized near the continuous outer membrane, suggestive for a non-destructive restructuring of the inner mitochondrial membrane. The smooth pinocytotic vesicles near the plasma membrane, lysosomes and heterogeneous dense bodies were more frequent. The revealed subcellular targets of BSH may initiate the development of pharmacological protocols aimed to further improve the tolerance to BSH by the healthy tissues of patients undergoing BNCT of brain tumors, e.g. by intervention into the oxidative stress triggered likely by the altered mitochondria.

Ultrastructure of nuclei of cisplatin-treated C6 glioma cells undergoing apoptosis.

C6 glioma cells, treated with a cytostatic dose of cisplatin (1.66 x 10(-5) M) ceased dividing by 24 h and, most of them had undergone apoptosis by 72-96 h. The reactive cells were classified into 5 types (T-I to V), according to the ultrastructure of nuclei. At 4 h, 20.4% of cells (T-I) showed minute condensation and margination of chromatin. The nuclear envelope (NE) formed slim and deep invaginations consisting of the inner or both membranes. The later kind of NE invaginations often extended to the enlarged nucleoli and contained nucleolus-like material at its cytoplasmic side. Some nuclear pores were covered with a dome-shaped "cap" formed by fine filamentous material. The number of T-I cells increased to 53.3% by 72 h. In T-II cells, which appeared at 24 h, the chromatin was condensed into dense irregular masses separated from the NE by a lucent space with filamentous structures preventing complete margination of chromatin. Nucleoli of T-II cells were small and showed partial segregation of their components. The "capped" pores were absent in these apparently more damaged cells. From 24 h, cells with large and lobulated nuclei (T-III) started to increase in number and peaked at 72 h (6.6%). Except for some small lobules, the chromatin of T-III cells was moderately aggregated and the NE was well preserved. Typical apoptotic cells with highly condensed and marginated chromatin (T-IV) peaked at 48-72 h (2.4-4.8%). They appeared in 2 varieties, including cells with wrinkled nuclei with less condensed and incompletely marginated chromatin or more lobulated forms with highly condensed marginated chromatin suggesting their origin from T-II or T-III cells. T-IV cells, as well as their fragments, underwent phagocytosis and secondary necrosis (T-V cells, 48.6% at 96 h). Two alternative routes of nuclear changes leading to cisplatin-triggered apoptosis, as represented by the sequence T-I --> T-III --> T-IV/V or T-I --> T-II --> T-IV/V, may explain the initially less or more damaged cells. These alternatives, together with progressive recruitment of reactive cells, suggest intrapopulation differences in the sensitivity of cells or in the cell cycle perturbations induced by cisplatin. Except for the T-IV and T-V cells, observed alterations of cytoplasmic organelles, including mitochondria, were fewer than reported in previous studies on cisplatin.

Ultrastructural response of the nuclear envelope (NE) of C6-glioma cells to cisplatin-induced apoptosis.

The early response of the nuclear envelope of C6-glioma cells (t < or = 24 h), treated with a cytostatic dose of cisplatin in culture (5 microg/ml) included formation of slim and deep invaginations formed by either the inner or both membranes. The invaginations made of the complete NE often extended up to the enlarged nucleoli. In some of them, nucleus-like material occurred at their cytoplasmic site suggesting its enhanced nucleus-to-cytoplasm transport. Some nuclear pores in the invagination-forming cells were covered by dome-shaped "caps" protruding into the karyoplasm. The "capped" pores were absent in the cells that were initially more damaged. At 48 to 72 h, we found a small number of large and hyperlobulated cells with some small lobules containing a rarefied chromatin and focally disintegrated NE. The lamina-free remnants of the NE with a swollen perinuclear cistern were still present at 72-96 h when the population entered the execution phase of apoptotic death.

Modulation of porphyrin derivatives accumulation in C6 glioma cells by drugs acting on beta-adrenergic receptors. A spectrofluorometric study.

The pharmacological modulation of the uptake of porphyrin derivatives in cultured C6 glioma cells was investigated by means of spectrofluorometric analysis both in single cells and in cell homogenates. The influence of drugs acting as beta-receptor agonists or antagonists was studied in cells grown to semiconfluency. Isoproterenol (ISO), a beta-receptor agonist, enhanced the intracellular fluorescence intensity of both Photofrin and protoporphyrin IX (PpIX). A treatment with a beta-receptor antagonist I-propranolol (PRO), simultaneous with ISO, resulted in an intracellular Photofrin fluorescence signal comparable to that of the control cells, indicating the specificity of the pharmacological action. The pharmacological treatment seemed particularly effective with the aggregated species. This is suggested by the relative increase of the band at 670 nm, being greater than that in the 630 nm band in the emission spectra of Photofrin and PpIX, and by the comparison of the fluorescence intensity on cell homogenates measured both in the absence and in the presence of cetyltrimethyl-ammonium bromide as a detergent.

Neurogenic control of the ovine gallbladder: ultrastructural and functional study.

The purpose of this study was to investigate the ultrastructure of the local nerve supply of ovine gallbladders as well as the functional characteristics of inhibitory nerves. We used electron microscopy of thin sections of ovine gallbladders and in vitro isometric tension recording using gallbladder strips. Specifically, we measured contractile and inhibitory responses induced by transmural electrical field stimulation (EFS). We found a ganglionated plexus with intramural nerve cells and interconnecting axons. Clear and large dense-core vesicles colocalized in axons close to smooth muscle cells. EFS elicited gallbladder contractions which were converted to relaxation after atropine. EFS-induced relaxation was reduced by the nitric oxide (NO) synthase inhibitor, L-NOARG and blocked by propranolol and/or tetrodotoxin. In conclusion, enteric ganglia and neurones with synaptic vesicles (clear and dense core) were detected close to smooth muscle bundles. Neural inhibition of gallbladder contraction was mediated by beta-adrenoceptors coupled to NO generation.

Ki-1-positive anaplastic large cell lymphoma initially diagnosed as Hodgkin's disease by fine needle aspiration (FNA) cytology.

A 45-year-old male presented with a large mass in the left axilla. FNA cytology was interpreted as Hodgkin's disease (HD), lymphocyte depletion (LD) type, but histopathologic and immunohistochemical examination showed features of Ki-1-positive anaplastic large cell lymphoma. Unrepresentative sampling by the FNA from the tumour periphery resulted in a false impression of dual reactive and neoplastic cell populations, which together with the frequent Reed-Sternberg-like cells led to the initial erroneous impression of HD. Therefore, the cytologic diagnosis of HD, LD should be approached with caution.

Renal aspergilloma due to Aspergillus flavus.

Renal aspergillomas have been reported only rarely. We report a case of Aspergillus flavus colonization of the renal pelvis and upper ureter of a patient with concomitant urinary schistosomiasis. The diagnosis was based on the demonstration of characteristic hyphal elements on direct microscopy and isolation of the fungus in culture. The patient was successfully treated with liposomal amphotericin B. This case emphasizes the importance of direct microscopic examination of urine specimens for prompt diagnosis of fungal infections of the urogenital system. Renal aspergilloma should be considered in the differential diagnosis of filling defects of the urinary tract, especially in patients who are immunocompromised.

Ultrastructure of human granulosa cells obtained from follicular fluid aspirates.

Samples of the granulosa cells obtained from a punctured human preovulatory follicles stimulated for in vitro fertilization purposes were studied by means of light and electron microscopic methods. Early and late preluteinized granulosa cells were characterized by the presence of high amount of pale and homogeneous lipid droplets, moderate accumulation of glycogene and progressive development of smooth endoplasmic reticulum. A new type of granulosa cell containing dense-cored granules with fine granular contents of a secretory nature was identified in this material and discussed in comparison with some earlier published indications of its possible existence.

Penetration of ovicidal fungus Verticillium chlamydosporium through the Ascaris lumbricoides egg-shells.

The course of penetration of the ovicidal fungus Verticillium chlamydosporium Goddard through the Ascaris lumbricoides egg-shells was studied in electron miorographs. The contact area between the egg and the fungus is smooth at the stage of the contact of the ovicidal fungus with the egg surface, as well as at the stage of adhesion. No special attaching organs are formed which indicates that no mechanical penetration of the fungal hypha through the egg-shells is involved. Traces of enzymatical activity of the fungus penetrating upright through the egg-shells can be observed during the penetration stage proper. At the consumption stage, the branching of the ovicidal fungus becomes more dense and some hyphae grow through the egg envelopes parallelly under the egg surface. The fungus grows through all parts of the infected egg, even through its contents.