Picosecond time-resolved infrared spectroscopic investigation into electron localisation in the excited states of Re(i) polypyridyl complexes with bridging ligands.

Mono- and binuclear complexes of (Re(CO)3Cl) with dipyrido[2,3-a:3',2'-c]-6,7-dimethylphenazine (ppbMe2) were synthesised and their photophysical properties probed using picosecond time-resolved infrared spectroscopy (TRIR). Excitation of these complexes in solution at 400 nm produces short-lived excited states. The IR spectrum of the excited state of the mononuclear [Re(CO)3Cl(ppbMe2)] have nu(CO) bands shifted to higher wavenumber relative to those of the ground state. This is consistent with formation of a (3)MLCT excited state. The IR spectrum of the excited state of the bimetallic [(Re(CO)3Cl)2(micro-ppbMe2)] shows the formation of two distinct groups of nu(CO) bands. This is interpreted as the formation of two distinct Re sites arising from a localised MLCT state with formally oxidised Re centre and a formally reduced bridging ligand. The nu(CO) bands of the adjacent Re centre are affected by the reduction of the bridging ligand. On the IR timescale the excited state structure is best formulated as [Cl(CO)3Re(II)(micro-ppbMe2 *-)Re(I)(CO)3Cl].     

Morphological and structural studies of early mineral formation in enamel of rat incisors by electron spectroscopic imaging (ESI) and electron spectroscopic diffraction (ESD)

Morphological and structural analysis of the earliest stage of crystal formation in enamel of rat incisors, by use of energy filtering transmission electron microscopy (EFTEM), has shown needlelike crystallites with a dotlike substructure. We conclude that these dots (nanometer-sized particles) have developed at nucleating, active sites along the non-collagenous matrix proteins in enamel. Calcium and phosphate groups are bound at such active sites and develop to nuclei, which grow to these stable dots (nanometer-sized particles). The dots coalesce rapidly in longitudinal direction, along the matrix proteins, with neighbouring dots to form parallel arranged needlelike crystallites. These needles grow and coalesce in lateral directions to ribbon-platelike crystallites. In enamel most of the organic substance becomes decomposed and transported to the ameloblasts. Consequently, the ribbon-platelike crystallites can coalesce to form much thicker (hydroxy)-apatite crystals than in dentine. Already in the earliest stage of crystal formation the mineral chains of dots (nanometer-sized particles) and the needlelike crystallites show a parallel orientation in the direction of the c-axis of hydroxyapatite. This is supported by the texture of the 002 reflections in the corresponding electron spectroscopic diffraction patterns (ESD), which appear as the first Bragg reflections.     

Taxol: an important new drug in the management of epithelial ovarian cancer.

Taxol, an antineoplastic agent isolated from the Pacific yew, has been demonstrated in three phase 2 clinical trials to have major activity (30 percent overall response rate) in patients with ovarian cancer refractory to cisplatin. The major toxicities associated with the agent are neutropenia (dose-limiting), hypersensitivity reactions, peripheral sensory neuropathy, and cardiac arrhythmias. A recently reported phase 1 trial of the combination of cisplatin and taxol has defined acceptable doses for the two-drug combination to be tested against cisplatin and cyclophosphamide as frontline therapy of advanced ovarian cancer. Taxol has also been examined for intraperitoneal administration in patients with ovarian cancer, with a major pharmacokinetic advantage for peritoneal cavity exposure being demonstrated. Unfortunately, any future development of taxol as an antineoplastic agent in the management of ovarian cancer will be dependent on the finding of an alternative source of the drug, as the current method of obtaining taxol from the bark of the Pacific yew provides insufficient quantities for large-scale clinical use.     

Antiproliferation effect of Rosemary (Rosmarinus officinalis) on human ovarian cancer cells in vitro

Rosemary (Rosmarinus officinalis L.) is a popular culinary/medicinal herb. Recent studies have shown it has pharmacologic activities for cancer chemoprevention and therapy. This study evaluated the antiproliferation activity of rosemary extract (RE) against human ovarian cancer cells, and whether the extract and its three main active ingredients carnosol (CS), carnosic acid (CA) and rosmarinic acid (RA) can enhance the antiproliferation activity of cisplatin (CDDP). Our study showed that RE has significant antiproliferation activity on human ovarian cancer A2780 and its CDDP resistant daughter cell line A2780CP70, with IC(50) (50% inhibitory concentration) estimated at 1/1000 and 1/400 dilutions respectively. RE enhanced the antiproliferation effect with CDDP on both A2780 and A2780CP70 cells. A2780 cells were consistently more sensitive to CS, CA, and RA than A2780CP70 cells between 2.5 and 20μg/ml. CS and RA also showed synergistic antiproliferation effect with CDDP on A2780 cells at some concentrations. RE treated by ultrafiltration, dialysis, and removal of phenolics lost the antiproliferation activity suggested that the activity resides in phenolics with MW<1000Da. Apoptosis array study of A2780 cells treated with RE showed that the expression of a number of genes regulating apoptosis were modulated by the treatment. This study showed that RE inhibited the proliferation of ovarian cancer cell lines by affecting the cell cycle at multiple phases. It induced apoptosis by modifying the expression of multiple genes regulating apoptosis, and holds potential as an adjunct to cancer chemotherapy.     

A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro.

To improve tumor-to-tissue ratios of anticancer agents in radioimmunotherapy, a three-step targeting approach was used to deliver biotinylated liposomes to human ovarian cancer cells (NIH:OVCAR-3, SK-OV-3) in vitro. Targeting was based upon the use of two antibodies specific for the CA-125 antigen that is highly expressed on NIH:OVCAR-3 cells but not expressed on SK-OV-3 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-CA-125 antibody and FITC-labeled streptavidin (SAv) prior to administration of biotinylated liposomes containing a marker dye for visualization by confocal laser scanning microscopy (CLSM). In addition, the two anti-CA-125 antibodies (B27.1 and B43.13) were labeled with FITC and incubated with ovarian cancer cells at 37 degrees C from 30 min to 24 h to study binding and uptake kinetics. Shedding kinetics of bound antibody from tumor cells was performed using radiolabeled B27.1. Results demonstrated that both B27.1 and B43.13 specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells. Biotinylation, FITC-labeling and radiolabeling of the antibodies did not compromise immunoreactivity. Less than 6% of the bound B27.1 was shed from tumor cells by 4 h following incubation, and the antibody-antigen complex resided predominantly on the cell surface by 4 h at 37 degrees C with slow internalization by 12-24 h. Biotinylated, conventional liposomes were specifically and effectively delivered to OVCAR-3 cells prelabeled with biotinylated B27.1 and SAv. The slow internalization and shedding properties of these antibodies are useful for multistep pretargeting methods. Thus, a modified targeting strategy, utilizing a bispecific antibody and liposomes, may be feasible for radioimmunoliposomal therapy of ovarian cancer.     

Refinement of the X-linked cleft palate and ankyloglossia (CPX) localisation by genetic mapping in an Icelandic kindred

The gene responsible for X-linked cleft palate and ankyloglossia (CPX) has previously been localized to the proximal region of the q arm of the X chromosome in both Icelandic and North American Indian kindreds. In this study, further linkage analysis has been performed on the Icelandic family and has resulted in a significant reduction in the size of the interval containing the mutated gene. A new polymorphism at DXS95, together with DXS1002 and DXS349, defines the proximal boundary of the CPX interval, whereas DXYS1X defines the distal boundary. Multipoint analysis supports this localisation with a peak lod score of 12.7, more than 2 lod score units higher than the next most likely position. In order to assess the physical size of the CPX interval prior to initiating yeast artificial chromosome cloning, metaphase fluorescence in situ hybridisation analysis was performed with the closest flanking markers. The size of the interval between DXS95 and DXYS1X was estimated to be approximately 2–3 Mb.     

Immunocytochemical localisation of PTHrP (parathormone-related peptide) in myoepithelial cells of human sweat and parotid glands.

PTHrP is a HHM-inducing peptide. It exhibits certain structural similarity to PTH and the two hormones may act through the same receptors. PTHrP is known to be produced in various tissues as well as during development. In this study we decided to immunocytochemically demonstrate PTHrP in normal skin and squamous cell carcinomas as well as in parotid glands (normal, inflamed and neoplastic). In the skin, PTHrP expression was demonstrated in epidermis and in smooth muscle cell layer of blood vessels. In squamous cell carcinomas, the expression was noted in foci of keratinization. In parotid glands, the peptide was localised in excretory ducts and in blood vessels, while inflammation of the gland and its tumours resulted most frequently in the less intense immunoreaction. The results are consistent with those of other authors. The novel observations include demonstration of PTHrP expression in myoepithelial cells of sweat glands and in parotid glands, where it may be involved in the control of their contractile activity.     

Examination of the phosphate in conjugative F-like pili by use of electron spectroscopic imaging.

F-like pili specified by conjugative plasmids have been reported to contain phosphate which may be noncovalently incorporated into the pilus. Electron spectroscopic imaging was able to detect phosphate in the filamentous, single-stranded DNA phage f1, used as positive control, but could not detect phosphate in F-like pili. Thus, the phosphate in purified pili which has been reported is probably derived from contaminating cell envelope material.     

Cellular localisation of the anti-cancer drug camptothecin in Camptotheca acuminata Decne (Nyssaceae)

In Camptotheca acuminata, we studied the cellular sites of accumulation of the alkaloid camptothecin (CPT), in both plants grown in the field and those grown in a greenhouse, subjecting the latter to stress (i.e., draught, nutritional deficit, and pruning). Fresh sections of the leaf, stem, and root were analysed for the presence of CPT by examining the autofluorescence that the CPT molecule emits when exposed to UV radiation. In the plants grown in the field, CPT was observed only rarely. In the greenhouse plants, CPT had accumulated in crystalline form in the vacuole of specialised cells (i.e., segregator idioblasts), which were not morphologically distinguishable from the cells of the surrounding tissues. In the organs examined, the segregator idioblasts were localised in parenchymatic and epidermal tissues. CPT crystals were also detected in the glandular trichomes on both the stem and leaf.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride. A study using electron spectroscopic imaging

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED = effective dose) and a toxic (LD25; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

Surfactant-like material on the chemoreceptorial surface of the frog's taste organ: an ultrastructural and electron spectroscopic imaging study.

Tannic acid treatment was used to study the morphology of surfactant-like material (SLM) in the taste organ of Rana esculenta and the relation between this material and the cell types of the organ. On the surface of the taste organ SLM was associated with the apical processes of wing and putative taste cells. In SLM, a biphasic pattern was visible, a portion showed a lamellar periodicity (the repeating period of lamellae approximated 45 A), and a second portion showed an homogeneous electron density. Electron spectroscopic imaging revealed the presence of phosphorus and a large amount of calcium associated with the SLM. The result of our work suggests that SLM has a role in the perireceptorial events in the gustatory transduction by concentrating calcium in specific sites of the chemoreceptorial surface.     

Endocrine cells of the APUD-system in the human lung (electron microscopy characteristics)

Lungs of 4 human fetuses (11-, 13-, 22-, 28-week-old), of 1 stillborn and of 3 mature persons, operated in connection with pulmonary cancer, have been investigated. In the fetal lungs apudocytes and neuroepithelial bodies (NEB) have been revealed. The apudocytes differ from each other by structure and size of endocrine granules. In the 11-week-old fetus P1 cells with two types of granules occur most often. Among P1 cells there are several subgroups, differing in their granule dimensions. P2 apudocytes possess granules of one type with a round core and a narrow rim of cytoplasm. P3 cells are characterized with still larger granules, a very dense core and a narrow rim. In large bronchi some groups are found, consisting of two and more endocrine cells of all three types. In the lungs of the 13-week-old fetus P1 cells are defined and a new type of cells, that contain homogenous granules, characterizing by their small size. In 22 weeks of development in the intrapulmonary bronchi apudocytes with granules specific for Ec-cells are found. NEB consists of cells and islands, possessing polymorphous granules. Various types of apudocytes are defined in large bronchi of the 22-week-old fetus. In the stillborn infant apudocytes in the lung are found very seldom. In lung of the mature persons the morphology of apudocytes is unitypical. Thus, during embryogenesis and after birth there are variable types of endocrine cells and NEB.     

Effect of glutathione depletion on antitumor drug toxicity (apoptosis and necrosis) in U-937 human promonocytic cells. The role of intracellular oxidation

Treatment with the DNA topoisomerase inhibitors etoposide, doxorubicin, and camptothecin, and with the alkylating agents cisplatin and melphalan, caused peroxide accumulation and apoptosis in U-937 human promonocytic cells. Preincubation with the reduced glutathione (GSH) synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO) always potentiated peroxide accumulation. However, although GSH depletion potentiated the toxicity of cisplatin and melphalan, occasionally switching the mode of death from apoptosis to necrosis, it did not affect the toxicity of the other antitumor drugs. Hypoxia or preincubation with antioxidant agents attenuated death induction, apoptotic and necrotic, by alkylating drugs. The generation of necrosis by cisplatin could not be mimicked by addition of exogenous H(2)O(2) instead of BSO and was not adequately explained by caspase inactivation nor by a selective fall in ATP content. Treatment with cisplatin and melphalan caused a late decrease in mitochondrial transmembrane potential (DeltaPsim), which was much greater during necrosis than during apoptosis. The administration of the antioxidant agents N-acetyl-l-cysteine and butylated hydroxyanisole after pulse treatment with cisplatin or melphalan did not affect apoptosis but attenuated necrosis. Under these conditions, both antioxidants attenuated the necrosis-associated DeltaPsim decrease. These results indicate that oxidation-mediated alterations in mitochondrial function regulate the selection between apoptosis and necrosis in alkylating drug-treated human promonocytic cells.     

Magnetic resonance imaging (MRI) in the diagnosis of recurrences of ovarian cancer in the small pelvis

The paper provides the results of small pelvic magnetic resonance tomography (MRI) in 62 patients with ovarian cancer after primary special treatment. Out of them 50 patients were found to have recurrences and metastases of the underlying disease, 12 patients had clinical remission. The study yielded MR signs and MR semiotics of recurrences of ovarian cancer in the small pelvis. The capacities of MRI with low and high intensities of a magnetic field were comparatively studied in the diagnosis of recurrences and metastases of ovarian cancer.     

Cellular and biomolecular responses of human ovarian cancer cells to cytostatic dinuclear platinum(II) complexes

Polynuclear platinum(II) complexes represent a class of potential anticancer agents that have shown promising pharmacological properties in preclinical studies. The nature of cellular responses induced by these complexes, however, is poorly understood. In this research, the cellular responses of human ovarian cancer COC1 cells to dinuclear platinum(II) complexes {[cis-Pt(NH₃)₂Cl]₂L¹}(NO₃)₂ (1) and {[cis-Pt(NH₃)₂Cl]₂L²}(NO₃)₂ (2) (L¹ = α,α′-diamino-p-xylene, L² = 4,4′-methylenedianiline) has been studied using cisplatin as a reference. The effect of platinum complexes on the proliferation, death mode, mitochondrial membrane potential, and cell cycle progression has been examined by MTT assay and flow cytometry. The activation of cell cycle checkpoint kinases (CHK1/2), extracellular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) of the cells by the complexes has also been analyzed using phospho-specific flow cytometry. Complex 1 is more cytotoxic than complex 2 and cisplatin at most concentrations; complex 2 and cisplatin are comparably cytotoxic. These complexes kill the cells through an apoptotic or apoptosis-like pathway characterized by exposure of phosphatidylserine and dissipation of mitochondrial membrane potential. Complex 1 shows the strongest inductive effect on the morphological changes of the cells, followed by cisplatin and complex 2. Complexes 1 and 2 arrest the cell cycle in G2 or M phase, while cisplatin arrests the cell cycle in S phase. The influence of these complexes on CHK1/2, ERK1/2, and p38 MAPK varies with the dose of the drugs or reaction time. Activation of phospho-ERK1/2 and phospho-p38 MAPK by these complexes is closely related to the cytostatic activity. The results demonstrate that dinuclear platinum(II) complexes can induce some cellular responses different from those caused by cisplatin.     

Localization of calcium and phosphorus in early predentin-matrix components by electron spectroscopic imaging (ESI)-analysis in rat molars

Summary The subcellular distribution of the inorganic elements calcium (Ca) and phosphorus (P) was studied in the first-formed dentin matrix during initial mineralization in neonatal rat molars. This most peripheral matrix region is comprised of a proteoglycan-rich ground substance, interwoven by a collagenous network, matrix vesicles, aperiodic fibrils derived from the dental basal lamina, and apical odontoblastic cell processes. All matrix components may possibly serve as templets for mineral deposition during initial calcification of first-formed mantle dentin and predentin. By means of the very sensitive ESI-analysis we studied the subcellular localization of Ca and P and their possible association with distinct organic extracellular matrix components and odontoblasts. Ca-signals were found in the ground substance, at striated collagen fibrils and plasma membranes of odontoblasts in the cuspal early matrix region, but occurred only sparsely in the ground substance of the more distal matrix region where odontoblast processes attach to aperiodic fibrils of the dental basal lamina. Ca was generally absent in matrix vesicles. In contrast, P-signals were found in matrix vesicles, at aperiodic fibrils and at the plasma membranes of odontoblasts. Ca and P co-localized at striated collagen fibrils (type I or II). These results suggest that striated collagen fibrils might serve as primary deposition sites for calcium phosphate during early biological calcification of organic extracellular macromolecules.     

Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells

Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.     

Primary ovarian cancer cell inhibition by human Wharton's Jelly stem cells (hWJSCs): Mapping probable mechanisms and targets using systems oncology

Ovarian cancer is one of the most lethal gynaecological cancers. Its subtle onset and absence of symptoms in early stages areassociated with poor prognosis and high mortality. Identification of early biomarkers would aid in ovarian cancer control.Mesenchmal stem cells (MSCs) and/or their secretory products are identified to have cancer inhibitory properties. Therefore, it isof interest to study the anticancer properties of human Wharton''s jelly stem cells conditioned medium (hWJSCs-CM) on primaryovarian carcinoma cells in vitro. Primary cultures of epithelial ovarian carcinoma cells (EOCs) and hWJSCs were used in thisstudy. EOCs were exposed to hWJSC-CM (100%) for 24h-72h and changes in mophology and cell proliferation were monitored.Treatment with hWJSC-CM showed altered morphological changes that resulted in death of EOCs. Colorimetric assay [MTT, (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)] showed mean decreases in EOC proliferation by 16.21%, 23.89% and40.08% at 24h, 48h and 72h respectively compared to control. Ingenuity Pathway Analysis (IPA, Igenuity Systems, USA) deducedimportant molecular pathways and signaling networks associated with cancer cell death and these correlated with significantexpression of tumour suppresors and apoptotic genes in hWJSCs. Secretory products of hWJSC-CM induced cell death of EOCs viaapoptosis. IPA identification of canonical genes/pathways involved in EOCs that overlap with hWJSCs tumour suppressors andapoptosis genes further support this hypotheis. Additional in vitro and in vivo studies are necessary to validate EOCs inhibitionwith hWJSC-extracts towards their mechanism of action.