Ferrocene-containing cationic lipids for the delivery of DNA: oxidation state determines transfection activity.

The ability of two redox-active, ferrocene-containing cationic lipids [11-(ferrocenylundecyl)trimethylammonium bromide (FTMA) and bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA)] to transfect mammalian cells was investigated. This study sought to determine the range of conditions over which these lipids were capable of transfecting cells and whether the oxidation state of the ferrocenyl groups in these materials could be used to influence the extent of transfection. Experiments conducted in the COS-7 cell line demonstrated that reduced and oxidized FTMA were substantially cytotoxic and did not transfect cells. Subsequent experiments conducted using BFDMA and reporter plasmids encoding enhanced green fluorescent protein (EGFP) and firefly luciferase demonstrated that BFDMA was able to transfect cells. However, the extent of transfection depended significantly upon both the concentration of BFDMA and the oxidation state of the lipid. Quantitative characterization of cytotoxicity and gene expression demonstrated that a window of concentration existed over which reduced BFDMA was non-cytotoxic and yielded high levels of transfection, but over which electrochemically oxidized BFDMA yielded very low (background) levels of transfection. Characterization of lipoplexes using dynamic light scattering demonstrated that reduced and oxidized BFDMA formed small aggregates (ca. 90 to 250nm) at concentrations of lipid ranging from 2 to 10 microM. Taken together, these results demonstrate that the oxidation state of BFDMA, which can be controlled electrochemically, can be used to control the extent of cell transfection. These results could form the basis of transfection procedures that exploit the redox behavior of ferrocene-containing lipids to achieve active spatial and temporal control over transfection using electrochemical methods.     

Neuroendocrine cells in Malassez epithelium and gingiva of the cat

Malassez epithelium has been designated as epithelial cell rests, the biological significance of which is still under debate. This study was designed to analyze Malassez epithelium for the presence of neuroendocrine cells. Gingival tissue was included as a positive control. Using immunohistochemistry, confocal and light microscopy, Malassez epithelium and gingival epithelium from mature cats (n=5) were examined for cells containing the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Both Malassez epithelium and the basal epithelial cell layers in gingival rete pegs regularly displayed cells immunoreactive to CGRP, SP, and VIP. The immunopositive cells were most frequently present in the epithelial cell clusters and strands of Malassez located in the cervical half of the periodontal ligament. Double immunolabeling revealed cellular co-expression of CGRP or SP with VIP, and the neuropeptides were co-localized in the cellular compartments. Labeled cells in both epithelia were occasionally supported by immunoreactive nerve fibers. This study shows that cells immunoreactive to CGRP, SP, and VIP are located within the cat Malassez epithelium. The localization of neuroendocrine cells verifies the diversity of this epithelium and confirms that Malassez epithelium is composed of different cell types, in common with epithelia from other locations. The presence of neuroendocrine cells in Malassez epithelium strongly suggests biological functions of this tissue, and the neuropeptide content may thus indicate endocrine functions of the cells.     

Enhancement of the antitumor efficacy of the antiprogestin,onapristone, by combination with the antiestrogen,ICI 164384

So far, no combination of endocrine treatments has been routinely used in the therapy of breast Cancer. It was, therefore, our interest to determine whether the combination of the antiprogestin, onapristone (ON), and the pure antiestrogen, ICI 164384 (ICI), might provide a more effective therapy than either monotherapy in experimental mammary tumors containing both estrogen and progesterone receptors. In the MXT-mammary tumor of the mouse, ON (5 mg/kg) administered for 3 weeks exerted an ovariectomy-like antitumor effect (56% inhibition), whereas ICI (30 mg/kg) was weakly effective (28% inhibition). The combination of ON and ICI was, however, distinctly more effective than the monotherapies or ovariectomy, causing 78% inhibition. A similar potentation of antitumor effect by the combination was manifested in the dimethylbenzanthracene-induced mammary tumor of the rat when ON (5 mg/kg) and ICI (30 mg/kg) were administered once daily for 4 weeks (s.c.). The remission rates of tumors found after treatment with ICI, ON, the combination and ovariectomy (complete and partial remission) were 15%, 46%, 71% and 100% respectively. In the animals bearing DMBA-induced tumors, treatment with ON alone significantly increased the serum levels of luteinizing hormone and prolactin, but caused only a slight increase in the peripheral levels of estradiol and progesterone. ON had no appreciable effect on the uterine and ovarian weights. ICI reduced the uterine weight and the serum progesterone level. In the combination with ON, ICI reversed the effect of ON on the progesterone level without influencing the luteinizing harmone and prolactin levels. These findings suggest that the augmentation of antitumor effectiveness by the combination of two antihormones can be ascribed not only to their effects at estrogen- and progester-one-receptor-binding sites, but also to the decrease in the peripheral level of progesterone. Thus, an appropriate combination of antiprogestin and pure antiestrogen may be useful in the management of breast cancer.     

The prognostic significance of overexpression of the decoy receptor for Fas ligand (DcR3) in patients with gastric carcinomas

Abstract. Background: The FasL-Fas system has an important role in mediating immune-cytotoxic killing of cells such as virus-infected or tumor cells. It was recently reported that there is a soluble decoy receptor (DcR3), which binds to FasL and inhibits FasL-induced apoptosis, and certain tumors may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL. We evaluated whether DcR3 has clinical relevance in actual human gastric cancers. Methods: . The expression of DcR3 was investigated by Northern blot analysis in a series of 84 primary gastric carcinomas and compared with clinicopathological features and prognosis. The DcR3 expression level was analyzed and quantified densitometrically. The location of DcR3 mRNA in gastric carcinoma tissue was detected by in situ hybridization. Results: The frequency of DcR3 overexpression was 26% (22 of 84 surgical specimens). The DcR3 expression level was significantly associated with lymph node metastasis and pathological stage, but did not correlate with tumor size, metastatic status, or histological type. In situ hybridization demonstrated that DcR3 mRNA was expressed in tumor cells. When the patients were followed up for 63 months, DcR3 overexpression was found to be associated with a significantly shortened duration of overall survival compared with findings in patients having normal DcR3 expression. Conclusion: The DcR3 decoy receptor for FasL may be involved in the progression of gastric cancer. Further evaluation of these possible roles of DcR3 and the regulation of DcR3 expression in malignant cells will be critically important for the development of new strategies for controlling the growth of malignant cells that escape host immune surveillance. Received: July 19, 2001 / Accepted: December 21, 2001     

beta(2)-Adrenoceptor activation attenuates endotoxin-induced acute renal failure

Abnormalities in the beta(2)-adrenergic control of organ function have been implicated in the pathogenesis of several disease states, such as septic shock. The objectives of the present study were to define the contribution of beta(2)-adrenoceptors (beta(2)-AR) to normal renal physiology and to investigate whether overexpression of renal beta(2)-AR might be potentially beneficial in preventing progressive renal damage associated with endotoxemia. Adenoviral transgenes containing the human beta(2)-AR (Adeno-beta(2)-AR) were constructed and delivered into the rat kidney by means of intraparenchymal injections. Administration of 10(9) total viral particles of Adeno-beta(2)-AR induced an approximately threefold increase in beta(2)-AR density in the renal tissue, which 2 wk after delivery, enhanced GFR and sodium reabsorption compared with control rats. The enhanced GFR was abolished by the addition of the beta(2)-AR antagonist, ICI 118,551. Administration of lipopolysaccharide (LPS) caused a reduction in GFR, beta(2)-AR density, and cAMP together with enhanced TNF-alpha mRNA in the kidney. In rats overexpressing beta(2)-AR, the reduction in baseline GFR and elevation of TNF-alpha mRNA and leukocyte infiltration into the kidney associated with the endotoxin were blocked. These findings suggested the possibility that a renal-specific overexpression of beta(2)-AR preserves basal renal function in response to a ligand-independent beta(2)-AR activation and that the delivery of Adeno-beta(2)-AR gene is a potential novel therapeutic strategy for treatment of acute renal failure associated with sepsis.     

Beta2-adrenoceptor activation inhibits Shiga toxin2-induced apoptosis of renal tubular epithelial cells

Apoptosis is regulated by several pathways, such as caspases, mitogen activated protein kinase (MAPK) and cAMP/cAMP-dependent protein kinase A (PKA) cascade. This study investigated the effect of beta(2)-adrenoceptor activation on Shiga toxin (Stx)2-induced apoptosis in renal tubular cells and the contribution of these signalling pathways. Cultured human adenocarcinoma-derived tubular cells were exposed to Stx2 (64 pg/mL) for 2-24hr following the addition of the beta(2)-adrenoceptor agonist (terbutaline) to the incubation medium. Stx2-induced apoptosis and its amelioration by beta(2)-adrenoceptor activation was confirmed using DNA degradation assays and by flow cytometry for annexin V, mitochondrial membrane potential and caspase(-3 and -7) activity. Exposure of cells to Stx2 for 24hr increased the DNA fragmentation to 11.6+/-0.9%, compared to 3.3+/-0.2% in control cells (P<0.05) but was decreased to approximately 5-7% (P<0.05) in the presence of terbutaline. Furthermore, Stx2-stimulated apoptosis, detected by TUNEL, annexin V and mitochondrial potential, was inhibited by terbutaline (P<0.05) which was prevented by cAMP-PKA inhibitors and a beta(2)-adrenoceptor antagonist. However, inhibition of Stx2-mediated caspase activity by terbutaline was partially blocked by cAMP-PKA inhibitors. On the other hand, p38MAPK inhibition by terbutaline prevented Stx2-induced apoptosis and caspase activity through a cAMP-independent pathway via beta(2)-adrenoceptor. These data indicate that beta(2)-adrenoceptor activation can inhibit Stx2-induced apoptosis of the cells, which may be caused by a reduction in caspase activity through cAMP-PKA activation and the p38MAPK pathway.     

Post-stroke treatment with imidapril reduces learning deficits with less formation of brain oedema in a stroke-prone substrain of spontaneously hypertensive rats

The present study was undertaken to examine the effects of the ACE (angiotensin converting enzyme) inhibitor imidapril, on the brain, when administered after the onset of stroke in a stroke-prone substrain of spontaneously hypertensive rats (SHRSP). Learning deficits and induced lesions in the brain as well as in the kidneys and heart were investigated in detail. SHRSP were divided into two groups with or without salt loading at the age of 4 weeks. The salt loading was performed for 7-9 weeks to increase the incidence of stroke. Within 24 h after the first observation of stroke, animals were subsequently treated with 5 mg/kg imidapril orally once a day or the vehicle for up to the age of 27 weeks. Imidapril attenuated progression of neurological abnormalities such as irritability, hyperkinesia and motor dysfunction, and increased survival rate. In three-panel runway testing, learning deficits did not develop significantly in the imidapril-treated group, and was comparable to that in the non-salt-loaded/non-stroke group. Imidapril reduced oedema formation in the cortex, hippocampus and striatum, and also suppressed lesion formation in the kidneys and heart. Imidapril thus suppressed progression of neurological deficits with loss of learning ability following onset of stroke, and also suppressed formation of oedema in the brain and decreased the number of lesions in other organs. Imidapril-induced reduction of cerebrovascular damage, which presumably occurs in the brain after stroke, may account for the inhibitory effects of imidapril on lesion formation and learning impairment.     

Effect of cyproterone acetate in comparison to flutamide and megestrol acetate on the ventral prostate, seminal vesicle, and adrenal glands of adult male rats

Cyproterone acetate (CPA) has a stronger inhibitory effect than megestrol acetate (MGA) and flutamide (FL) on the prostate and seminal vesicle of intact adult rats. Only in the clinically irrelevant regimen of castration and simultaneous androgen substitution is FL as a competitive androgen antagonist more potent than CPA and MGA. The latter inhibit complete involution of the ventral prostate but not of the seminal vesicle of castrated adult rats. This effect is very small in magnitude, cannot be increased by the use of higher doses, and is only reduced but not blocked by simultaneous treatment with high doses of FL. Further, CPA is unable to stimulate proliferation or restore the function of the involuted rat prostate. CPA and MGA inhibit adrenal weight in rats, thus indicating a glucocorticoidlike activity in this species. A critical review of all available data on the antiandrogenic, glucocorticoidlike, and possible paradoxical androgenlike activities of CPA in different animal experiments has no important bearing on the clinical effectiveness of CPA in the treatment of prostatic cancer.     

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons were investigated by an immuno-cytofluorometric method. The receptor density showed a drastic decrease around birth and a smaller change after birth.