Fenugreek improves diet-induced metabolic disorders in rats.

Trigonella foenum-graecum L. (fenugreek) has been described earlier and its use in ancient medicinal practice is well known. The hypoglycemic effects of fenugreek have been studied in many animal models and diabetic patients. The purpose of this study was to examine the preventive efficiency of dietary fenugreek on diet-induced metabolic diseases in rats. The diets used in this study were a standard diet, a high-fat high-sucrose (HFS) diet, and a HFS diet containing 0.5 g/kg b. w./day fenugreek based on the modified version of the AIN-93G purified diet, for 12 weeks, respectively. The rats fed the HFS diet containing fenugreek showed significantly lower fasting insulin levels and HOMA-IR than the rats fed the HFS diet. Therefore, fenugreek improved insulin sensitivity in rats. The triglyceride and total cholesterol levels in the plasma were significantly lower in the fenugreek-administered group. Moreover, distinct reductions of triglyceride, total cholesterol, free fatty acid, and phospholipid levels in the liver were found in the rats fed the HFS diet containing fenugreek. These results suggest that fenugreek enhanced insulin sensitivity at least partly by improving lipid metabolism disorders in the plasma and the liver in the rats induced by the HFS diet.

Vasopressin-dependent upregulation of aquaporin-2 gene expression in aged rats with glucocorticoid deficiency.

AIM: The study was undertaken to determine whether ageing affects kidney expression of the aquaporin-2 (AQP2) water channel in glucocorticoid-deficient rats. METHODS: After adrenalectomy, 6- and 52-week-old Sprague-Dawley rats received aldosterone via osmotic minipumps (glucocorticoid-deficient rats). Aldosterone and dexamethasone were administered to control rats of the same age. RESULTS: An acute water load test verified impairment of water excretion in both young and aged rats with glucocorticoid deficiency, with a more serious impairment in the older rats. Despite the presence of hypoosmolality, non-suppressible release of arginine vasopressin (AVP) was particularly evident in the aged rats with glucocorticoid deficiency in comparison with the young rats. The expression levels of AQP2 mRNA and protein were lower in the aged rats, with a particularly large reduction in AQP2 protein expression. AQP2 expression levels were significantly augmented in the glucocorticoid-deficient rats compared with the controls under both basal and water-loaded conditions. Acute water loading did not suppress expression of AQP2 mRNA and protein, and the percentage increases in AQP2 mRNA and protein expression vs. the respective controls were more pronounced in the 52-week-old glucocorticoid-deficient rats compared with the 6-week-old rats. CONCLUSION: The findings indicate that upregulation of AQP2 expression is maintained dependent upon non-suppressible release of AVP in rats with glucocorticoid deficiency, and that AQP2 plays a crucial role in persistent impairment of water excretion in aged rats with glucocorticoid deficiency.

Nicorandil improves diabetes and rat islet beta-cell damage induced by streptozotocin in vivo and in vitro.

OBJECTIVE: N-(2-hydroxyethyl)-nicotinamide nitrate (nicorandil) is a unique anti-anginal agent, reported to act as both an ATP-sensitive K(+) channel opener (PCO) and a nitric oxide donor. It also has an anti-oxidant action. We examined the effects of nicorandil on streptozotocin (STZ)-induced islet beta-cell damage both in vivo and in vitro. DESIGN AND METHODS: STZ-induced diabetic Brown Norway rats (STZ-DM) were fed with nicorandil-containing chow from day 2 (STZ-DM-N48), 3 (STZ-DM-N72), and 4 (STZ-DM-N96) to day 30. Body weight, blood glucose, and plasma insulin were measured every week. For the in vitro assay, neonatal rat islet-rich cultures were performed and cells were treated with nicorandil from 1 h before to 2 h after exposure to STZ for 30 min. Insulin secretion from islet cells was assayed after an additional 24 h of culture. We also observed the effect of nicorandil on the generation of reactive oxygen species (ROS) from rat inslinoma cells (RINm5F). RESULTS: Body weight loss and blood glucose levels of STZ-DM-N48 rats were significantly lower than those of STZ-DM rats. Immunohistochemical staining of insulin showed preservation of insulin-secreting islet beta-cells in STZ-DM-N48 rats. Nicorandil also dose-dependently recovered the insulin release from neonatal rat islet cells treated with STZ in in vitro experiments. Nicorandil did not act as a PCO on neonatal rat islet beta-cells or RINm5F cells, and did not show an inhibitory effect on poly(ADP-ribose) polymerase-1. However, the drug inhibited the production of ROS stimulated by high glucose (22.0 mmol/l) in RINm5F cells. CONCLUSIONS: These results suggested that nicorandil improves diabetes and rat islet beta-cell damage induced by STZ in vivo and in vitro. It protects islet beta-cells, at least partly, via a radical scavenging effect.

Pathological role of aquaporin-2 in impaired water excretion and hyponatremia.

In the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), inappropriately elevated secretion of vasopressin can result in a reduction of antidiuretic efficacy: a phenomenon known as 'vasopressin escape'. We compared experimental SIADH with 1-deamino-8-d-arginine vasopressin (dDAVP)-excess rats, where both groups received continuous subcutaneous administration of dDAVP by osmotic minipump but the SIADH rats also received a liquid diet that induced hyponatraemia. The SIADH rats, but not the dDAVP excess rats, showed a marked attenuation of urinary concentrating ability. Vasopressin V(2) receptor binding capacity and mRNA expression were similar between the two groups, but the SIADH rats showed a diminished up-regulation of aquaporin-2 (AQP-2) mRNA and protein expression. These findings indicate the presence of tonicity-response regions in the AQP-2 promoter gene, and that either hypervolemia or hypotonicity may attenuate the postreceptor signalling of vasopressin in renal collecting duct cells in SIADH rats.

Overcoming multi-drug resistance using an intracellular anti-MDR1 sFv.

We made an intracellular single-chain variable fragment (sFv) from the C219 monoclonal antibody that recognized the intracellular domain of the multidrug resistance (MDR) gene product, P-glycoprotein (P-gp). Immuno-cytochemistry using the FITC conjugated anti-C-myc tag antibody showed that the sFv protein was expressed in the cytoplasm of the cells. Although transfection of the sFv did not result in the down-regulation of P-gp expression in P-gp positive MDR cells as determined by flow cytometry analysis, Adriamycin (ADM) uptake and Rhodamine123 (Rh123) retention were increased by the C219 intra-cellular sFv transfection. The transfected cells exhibited a higher sensitivity to ADM using a 10-day colony formation assay. The conventional 3-day MTT assay showed the drug resistant tendency in C219 sFv transfected cell we tested. The growth rate of C219 sFv transfected cells was delayed in all non-MDR and MDR cells that might be the reason why C219 transfected cells exhibited the drug resistant tendency in the MTT assay. Despite this unexpected effect of C219 sFv on growth rate, our data suggest that the intra-cellular sFv technique could knockout MDR functionally and may offer a means of increasing the effectiveness of tumor chemotherapy.

Cross-reactive mechanism for the false elevation of free triiodothyronine in the patients treated with diclofenac.

We report three cases of patients exhibiting a false elevation of serum free triiodothyronine (FT3) as a result of a cross-reaction with diclofenac. The first case is a 66-yr-old woman with a long history of rheumatoid arthritis (RA). The patient was receiving diclofenac for the treatment of her RA. The patient was subsequently diagnosed as having thyroid papillary adenocarcinoma and received a subtotal thyroidectomy. After the operation, the patient exhibited postoperative hypothyroidism except for a gradual elevation of FT3. The other two patients also exhibited an elevated serum FT3 level after the administration of diclofenac. Serum FT3 levels in these patients decreased to normal or below normal after diclofenac administration was discontinued. In the first case, the elimination of immunoglobulin from the sera using polyethylene glycol precipitation did not reduce the FT3 level. In our hospital, Vitros ECi (enhanced chemiluminescence enzyme immunoassay) system and Vitros FT3 kit were used for FT3 assay. The patient's FT3 levels were normal or below normal when they were measured using other FT3 kits. FT3 was also detected when diclofenac was dissolved in a phosphate buffered saline. Therefore, we concluded that a cross-reaction between FT3 and diclofenac was the mechanism causing the false elevation of FT3 in these patients.

Tetracycline-induced expression of an anti-c-Myb single-chain antibody and its inhibitory effect on proliferation of the human leukemia cell line K562.

Ablation of c-Myb function might be an effective approach for the therapy of chronic myelogenous leukemia or other c-myb-dependent malignancies. To this end, we have previously used an intracellular anti-c-Myb single-chain antibody (sFv) to achieve the functional knockout of the c-Myb oncoprotein. In this study, we have employed a tetracycline-inducible system to control the expression of the sFv. A nuclear-localizing form of an anti-c-Myb sFv was cloned into a tet-regulated plasmid vector. Using a transient expression system in COS-1 cells, we observed that doxycycline (Dox) induced expression of the sFv in a dose-dependent manner, and that the sFv was localized mainly in the nucleus. The Dox-induced anti-c-Myb sFv also inhibited the transactivating activity of c-Myb in a dose-dependent manner. We subsequently confirmed the Dox-induced expression of the sFv in the leukemia cell line K562. Proliferation of the target leukemia cells was also inhibited. These results suggest that the anti-c-Myb sFv may represent a viable method for gene therapy of c-myb-dependent hematopoietic malignancies.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy.

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.

Functional knock-out of c-myb by an intracellular anti-c-Myb single-chain antibody.

Aberrant expression of the c-myb proto-oncogene is a key factor in the development of the neoplastic phenotype in a variety of contexts. On this basis, it has been proposed that ablation of c-myb function might be an effective approach for therapy. To this end, we have employed an intracellular single-chain antibody (sFv) approach to achieve the functional knock-out of the c-Myb onco-protein. We derived an anti-c-Myb sFv, which was configured into eukaryotic expression plasmids. We confirmed the expression of the cytoplasmic and nuclear forms of the sFvs in the correct subcellular compartments by immunofluorescent staining. Importantly, the anti-c-Myb sFvs strongly inhibited the transactivation activity of c-Myb. Furthermore, cytotoxic effect of the sFv was observed only in the c-Myb positive cell line K562. These results suggest that anti-c-Myb sFv is a valuable tool for understanding the molecular mechanisms of c-myb induced transformation. In addition, this approach may have potential utility in the gene therapy for c-myb-dependent malignant diseases.

Phenotypic knock-out of the latent membrane protein 1 of Epstein-Barr virus by an intracellular single-chain antibody.

Epstein-Barr virus (EBV) causes lymphoproliferative diseases in immunocompromised patients and is associated with endemic Burkitt lymphoma, nasopharyngeal carcinoma and some cases of Hodgkin disease. The latent membrane protein 1 (LMP1) of EBV is a transmembrane protein that is essential for the transformation of B lymphocytes. LMP1-mediated up-regulation of Bcl-2 is thought to be an important element in this process. As an approach to explore novel treatments for EBV-associated lymphomas, we constructed a single-chain antibody (sFv) directed against LMP1 to achieve functional inhibition of this oncoprotein in EBV-transformed B lymphocytes. We demonstrated that intracellular expression of an endoplasmic reticulum (ER)-targeted form of this sFv markedly reduced LMP1 protein levels. We also observed a decrease in intracellular level of this protein which correlated with a marked reduction of Bcl-2 expression in EBV-transformed B lymphocytes. We further demonstrated that anti-LMP1 sFv-mediated reduction of Bcl-2 correlated with increased sensitivity of these cells to drug-induced cell death. Therefore, these data suggest that an anti-LMP1 sFv used in combination with conventional chemotherapy may be useful for gene therapy of EBV-associated lymphomas in immunocompromised patients.

Gene therapy strategies for AIDS-related malignancies.

AIDS-related malignancies (ARM) include AIDS-defining cancers such as Kaposi's sarcoma, non-Hodgkin's lymphoma and cervical carcinoma. In addition, certain other malignancies are also increased with human immunodeficiency virus (HIV) infection. New antiretroviral agents and better prophylaxis and treatment of HIV-related opportunistic infections are prolonging the lives of HIV-infected individuals. There will thus likely be a continued rise in the incidence and prevalence of ARM in the long term, even if effective antiretroviral and other AIDS-related therapies reduce their appearance in the short term. There are presently no curative regimens for the common ARM, with the possible exception of some lymphomas. Survival is shortened by most, and treatment is often toxic and poorly tolerated. Gene therapies may thus offer a useful adjunct to conventional treatment strategies for selected ARM. Although some gene therapy strategies may work well in the HIV setting, the chronic viral infection, immunodeficient status of the host, the tendency for HIV-infected individuals to have altered drug metabolism and an increased rate of adverse drug reactions will likely present special challenges. This review summarizes the state-of-the-art in the fledgling field of gene therapy for ARM, and explores areas for future research.

Appropriate intravenous doses of L-thyroxine and magnesium in a thyroidectomized patient with thyroid and parathyroid carcinomas receiving total parenteral nutrition during acute necrotizing pancreatitis.

A totally thyroidectomized patient with thyroid and parathyroid carcinomas, which had developed after neck irradiation in childhood, became hypercalcemic due to pulmonary metastases. The hypercalcemia was ameliorated by intermittent iv administration of bisphosphonate for 3.5 years, but this gradually became refractory to the bisphosphonate treatment. After right thoracotomy for resection of pulmonary metastases, acute necrotizing pancreatitis developed. The patient was therefore placed on total parenteral nutrition supplemented with T4 and a restricted dose of magnesium. Thyroxine(T4) (30 micrograms/day, iv) was not sufficient to maintain euthyroidism, but a higher dose (60 micrograms/day) elicited mild hyperthyroidism to the same extent as that elicited by an oral dose of 100 micrograms/day. The present case showed that the appropriate iv dose of T4 in this thyroidectomized patient with acute pancreatitis was about 60% of the oral dose. Furthermore, bisphosphonates (pamidronate and alendronate) and magnesium depletion were very effective in controlling the hypercalcemia.

Suppression of LTD in cultured Purkinje cells deficient in the glutamate receptor delta 2 subunit.

Involvement of the glutamate receptor channel delta 2 subunit in cerebellar long-term depression (LTD) was studied in cultures prepared from wild-type and mutant mice deficient in the delta 2 subunit. LTD of the glutamate response was induced by pairing glutamate applications and depolarization of a Purkinje cell in wild-type culture. However, in cultured Purkinje cells prepared from mutant mice, the same conditioning failed to induce LTD. Immunocytological staining showed that mutant Purkinje cells develop and express calbindin (a marker protein for Purkinje cells) as do wild-type cells, but they express no delta 2 subunit protein. The results indicate that the glutamate receptor channel delta 2 subunit is involved in the postsynaptic down-regulation of glutamate sensitivity, presumably during cerebellar LTD.

Involvement of inositol trisphosphate in cerebellar long-term depression.

We examined the role of increases in Ca2+ from different sources in the induction of long-term depression (LTD) of glutamate or AMPA responsiveness in cultured Purkinje neurones. Photolysis of caged Ca2+ or caged inositol 1,4,5-trisphosphate (InsP3) as well as depolarization was used to increase Ca2+ concentration. Heparin, contained in a patch pipette to block InsP3 binding to its receptor, prevented LTD induction by coupling of glutamate application and depolarization. Although pairing of depolarization and AMPA application did not induce LTD, photolysis of caged InsP3 in conjunction with depolarization and AMPA application induced LTD. The results suggest that not only Ca2+ influx through voltage-gated Ca channels but also InsP3-induced Ca2+ mobilization are involved in LTD induction.

Critical role of postsynaptic calcium in cerebellar long-term depression.

It has been proposed that postsynaptic Ca2+ is required for induction of cerebellar long-term depression (LTD). However, whether a depolarization independent increase in Ca2+ is sufficient for LTD induction remains to be determined. We used nitr-5, a photolabile Ca2+ chelator, to address this issue. Photolysis of nitr-5 together with glutamate application, but neither photolysis of nitr-5 alone nor glutamate application alone induced LTD. When two independent glutamate pipettes were aimed at different dendrites of the same Purkinje neurone, LTD induction was confined to the site at which glutamate and photolysis of nitr-5 were applied. These results indicate that activation of glutamate receptors together with intracellular Ca2+ increase without depolarization is sufficient to induce LTD in cultured Purkinje neurones.

Involvement of the glutamate receptor delta 2 subunit in the long-term depression of glutamate responsiveness in cultured rat Purkinje cells.

An antisense oligonucleotide against the glutamate receptor delta 2 subunit mRNA, which is selectively expressed only in Purkinje neurons, suppressed the induction of long-term depression (LTD) of glutamate responsiveness in the rat cerebellar culture. LTD of glutamate response is induced by pairing glutamate application and depolarization of a Purkinje cell. Treatment of the culture with the antisense oligonucleotide exerted no appreciable effect on basic physiological and morphological properties of Purkinje cells, except for LTD induction and reduction of delta immunoreactivity which was intense in distal dendrites. Sense and missense oligonucleotides, which were used as controls, did not block LTD induction. These results suggest that the glutamate receptor delta 2 subunit is involved in the cerebellar LTD.

Interleukin-4 inhibits spontaneous and parathyroid hormone-related protein-stimulated osteoclast formation in mice.

We examined the in vivo effects of recombinant murine IL-4 (rmIL-4) on spontaneous and stimulated mouse osteoclast formation. EC-GI cells, which produce PThrP and IL-1 alpha, were explanted in nude mice. These EC-GI cell-bearing nude mice developed hypercalcemia (4.90 +/- 0.68 mM), and the calcium levels were decreased to near normal (3.48 +/- 0.73 mM, p < 0.05) at day 3 by continuous infusion of rmIL-4 at a dose of 7 micrograms/day. When infused with 0.6 nmol/day of PTHrP(1-34) in ICR mice, rmIL-4 at a dose of 1 or 5 micrograms/day for 3 days caused a marked inhibitory effect on hypercalcemia induced by PTHrP(1-34) (3.73 +/- 0.56-2.54 +/- 0.14 mM, p < 0.01). However, rmIL-4 alone did not change the serum calcium in mice. Histomorphometric analysis revealed that rmIL-4 inhibits both spontaneous and PTHrP(1-34)-stimulated osteoclast formation in mice, with a decrease in osteoclastic surface and in the number of osteoclasts per mm bone surface, respectively. We conclude that IL-4 inhibits spontaneous and stimulated bone resorption resulting from inhibition of osteoclast formation and modulates the development of humoral hypercalcemia of malignancy.

Spatial distribution of excitatory and inhibitory synapses on a Purkinje cell in a rat cerebellar culture.

1. The spatial distribution of excitatory and inhibitory synapses on cultured Purkinje cells was studied with fluorescence, scanning electron microscopy (SEM), and electrophysiological techniques. 2. Presynaptic terminals were identified with immunohistochemical staining of synaptophysin and the results were correlated with SEM micrographs. 3. Excitatory and inhibitory inputs onto the Purkinje cell were identified from the direction and pharmacology of the postsynaptic current. 4. The localization of the presynaptic terminals on the Purkinje cell was observed after electrophysiological identification by filling the presynaptic neuron with Lucifer yellow and the Purkinje cell with Texas red. 5. The axon and presynaptic terminals of excitatory and inhibitory inputs had a different spatial organization. Excitatory inputs from granule cells were exclusively localized on the dendrites of Purkinje cells, whereas inhibitory contacts were found on both the soma and dendrites. This result is similar to that described in vivo.