Characterization of the putative cholesterol transport protein metastatic lymph node 64 in the brain.

Intracellular management of cholesterol is a critical process in the brain. Deficits with cholesterol transport and storage are linked to neurodegenerative disorders such as Neimann-Pick disease type C and Alzheimer's disease. One protein putatively involved in cholesterol transport is metastatic lymph node 64 (MLN64). MLN64 localizes to late endosomes which are part of the cholesterol internalization pathway. However, a detailed pattern of MLN64 expression in the brain is unclear. Using immunocytochemical and immunoblot analyses, we demonstrated the presence of MLN64 in several tissue types and various regions within the brain. MLN64 immunostaining in the CNS was heterogeneous, indicating selective expression in discrete specific cell populations and regions. MLN64 immunoreactivity was detected in glia and neurons, which displayed intracellular labeling consistent with an endosomal localization. Although previous studies suggested that MLN64 may promote steroid production in the brain, MLN64 immunoreactivity did not colocalize with steroidogenic cells in the CNS. These results demonstrate that MLN64 is produced in the mouse and human CNS in a restricted pattern of expression, suggesting that MLN64 serves a cell-specific function in cholesterol transport.

Oxysterols regulate expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20alphaOHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20alphaOHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.

Specific dose-dependent effects of ethane 1,2-dimethanesulfonate in rat and mouse Leydig cells and non-steroidogenic cells on programmed cell death.

The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively kills Leydig cells is poorly understood. To characterize further the cell-specific actions of EDS, we studied biochemical and morphological changes during apoptosis in different Leydig cell and non-steroidogenic cell models. Rat testicular and H540 tumor Leydig cells were killed by 1-2 mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher concentration of EDS was also necessary for activation of apoptosis in non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney cells were resistant. These variable effects of EDS on apoptosis were independent of new protein synthesis and, interestingly, could be delayed by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also observed chromosomal fragmentation and other hallmarks indicative of apoptosis as evidenced by DNA laddering and fluorescent microscopy. Time-lapse photography with a confocal microscope showed that the time of onset, duration and even the sequence of apoptotic events between individual H540 cells was heterogeneous. When the dose of EDS was gradually increased from 2 to 10 mM, the proportion of cells showing normal apoptotic features gradually decreased. Intriguingly, treatment with 10 mM EDS did not result in death for most cells and was marked by an absence of DNA laddering and ultrastructural features of apoptosis and necrosis. However, incubation with 20 mM EDS resulted in necrosis.These results demonstrated that the effects of EDS on cell survival are not specific to Leydig cells, that different cell types have different sensitivities to EDS and that stimulation of the cAMP pathway may mitigate EDS action. The data obtained with H540 cells further revealed that EDS can induce two types of programmed cell death.

Implications of progesterone metabolism in MA-10 cells for accurate measurement of the rate of steroidogenesis.

In virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period of 3 h. Steroids were then extracted, separated by HPLC, and identified by GC/MS. We found that more than 70% of radiolabeled steroids were converted to at least five different metabolites. A major metabolite (40%) was 5 alpha-pregnan-3 alpha or 3 beta-ol-20one. Similar studies, using radiolabeled T, demonstrated conversion to dihydrotestosterone and two forms of 5 alpha-androstane-diols. These data indicate the presence of active 5 alpha-reductase and 3 alpha- and/or 3 beta-hydroxysteroid dehydrogenase activities in MA-10 cells. Because these results suggest that progesterone is an unstable end product, to gauge the level of active metabolism, we incubated cells in the presence of inhibitors of pregnenolone metabolism and assessed pregnenolone levels by RIA. We discovered that basal levels of steroidogenesis in MA-10 cells were considerably higher than previously estimated. Moreover, dibutyryl cAMP-stimulated steroid production was linear over more than 13 h, in contrast to previous findings that measured progesterone levels. Other consequences of inaccurate assessment of steroidogenic activity in MA-10 cells because of the application of the progesterone assay are discussed.

Preclinical development of the Islet Sheet.

The Islet Sheet is a thin planar bioartificial endocrine pancreas fabricated by gelling highly purified alginate and islets of Langerhans. Acellular alginate layers form a uniform immunoprotective barrier to host rejection of the encapsulated cells, with the tissue nourished by passive diffusion from adjacent host tissue. The overall thickness of the Islet Sheet, 250 microm, is chosen to maximize nutrient diffusion. In this paper we describe the early development of the Islet Sheet, including purification and fractionation of the alginates used, difficulties in maintaining sheet planarity, and preliminary metabolic studies in pancreatectomized dogs. In a key experiment, approximately 75,000 allogeneic islet equivalents in six Islet Sheets were sutured to the omentum of a 7-kg female beagle dog at the time of pancreatectomy. Fasting euglycemia was maintained for 84 days. Fed blood sugars were usually below 150 mg/dL. A single injection of 2 U insulin was administered on day 9, and antibiotics were administered for two weeks. No other drugs were used. IVGTT post implant was not normal, but seemed to improve between 30 and 60 days. Upon omentectomy and sheet removal the metabolic parameters deteriorated to a frankly diabetic state within seven days. The sheets did not remain flat, but fragments were recovered within hard, mostly acellular capsules. Dithizone staining showed islets within alginate sheets recovered from the interior of these capsules, suggesting that allogeneic islet tissue survived 84 days and was responsible for maintaining fasting euglycemia.

Novel antihyperglycemic terpenoid-quinones from Pycnanthus angolensis.

Two new compounds, pycnanthuquinone A (1) and pycnanthuquinone B (2), were isolated from leaves and stems of the African plant, Pycnanthus angolensis (Welw.) Warb (Myristicaceae), by bioassay-guided fractionation of an ethanolic extract using a diabetic mouse model. Pycnanthuquinones A and B are the first representatives of a novel terpenoid-type quinone skeleton, and both compounds possess significant antihyperglycemic activity.

Nigericin inhibits accumulation of the steroidogenic acute regulatory protein but not steroidogenesis.

The steroidogenic acute regulatory (StAR) protein mediates the delivery of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol side chain cleavage complex converts it to pregnenolone. While the mechanism by which this mitochondrial protein acts is poorly understood, one component of the mitochondrial electrochemical gradient, the electrochemical potential (DeltaPsi), appears to be essential. In this study, the importance of the other component, the proton gradient (DeltapH), was examined. Disruption of DeltapH with the electroneutral K(+)/H(+) exchanger, nigericin, had no effect on steroidogenesis in MA-10 mouse Leydig tumor cells at concentrations which significantly reduced StAR protein levels. These data indicate for the first time in true steroidogenic cells, that StAR can act prior to being fully imported into the mitochondria and are consistent with observations made in COS-1 cells using mutant forms of StAR. These results support the hypothesis that a DeltaPsi-dependent factor is required for StAR activity and demonstrate that nigericin is the first compound described, capable of inhibiting StAR accumulation without affecting steroidogenesis.

Antihyperglycemic acetylenic glucosides from Bidens pilosa.

In vivo bioassay-guided fractionation of the aqueous alcohol extract of the aerial parts of Bidens pilosa Sch. Bip. var. radiata (Asteraceae) using C57 BL/Ks-db/db mice as a model for type 2 diabetes, yielded two known polyacetylenic glucosides, identified as 2-beta-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-+ ++triyne (1) and 3-beta-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,1 2-triyne (2). A 3:2 mixture of compounds 1 and 2 effected a significant drop in blood glucose.

Antihyperglycemic activity of Teramnus labialis (Fabaceae).

In vivo bioassay-guided fractionation of the aqueous alcohol extract of the aerial parts of Teramnus labialis (Roxb.) Benth. (Fabaceae), using C57BL/Ks-db/db mice as a model for type 2 diabetes, yielded an active fraction containing a mixture of coumarins. The major coumarin present in the active fraction was identified as fraxidin.

Transplantation of normal and genetically modified adrenocortical cells.

Cell transplantation techniques have been applied to the study of the biology of the adrenal cortex and to adrenocortical cell proliferation, differentiation, and senescence. Primary bovine adrenocortical cells, primary human adrenocortical cells and genetically modified bovine adrenocortical cells have been transplanted. Successful methods include transplantation of cells beneath the kidney capsule and several subcutaneous cell transplantation procedures. In successful transplants the cells form a functional vascularized tissue structure that allows the host animals to survive adrenalectomy. We show here that subcutaneous cell transplantation does not depend on embedding cells in collagen gel before introduction into the host animal. Subcutaneous transplants secrete both cortisol and aldosterone. However, the variability of plasma aldosterone levels indicates that the factors that determine glomerulosa-type and fasciculata-type cell function in transplant tissues are not well understood.

Effect of poststroke captopril treatment on mortality associated with hemorrhagic stroke in stroke-prone rats.

We tested the ability of captopril treatment (50 mg/kg/day p.o.), initiated 2 weeks before stroke or up to 5 days after stroke, to alter the onset of stroke and death after stroke in Kyoto Wistar stroke-prone spontaneously hypertensive rats (SHRsp). The benefits of blood pressure and aldosterone suppression during captopril treatment were assessed. SHRsp developed a 100% mortality rate with intracerebral hemorrhage by 16 weeks of age. Captopril treatment, started 2 weeks before or at the initiation of stroke, suppressed plasma aldosterone and equally prevented mortality to a mean age of >27 weeks. Treatment started 5 days after stroke extended the mean lifespan to >23 weeks. The re-elevation of plasma aldosterone (via osmotic pumps to levels in untreated SHRsp) during captopril treatment, before stroke, allowed stroke to develop. The initiation of the latter manipulation in pre- or poststroke captopril-treated SHRsp at a latter age (23 weeks) didn't alter the lifespan of SHRsp (death occurred at about 28 weeks). The antistroke effects of captopril treatment occurred without an antihypertensive effect, weren't altered by enhancing hypertension during treatment (with dexamethasone), and couldn't be duplicated by antihypertensive treatment with hydralazine. Spironolactone treatment didn't duplicate the effects of captopril. The suppression of plasma aldosterone may retard the onset of stroke in SHRsp during captopril treatment but likely other factors prolong life in pre- and poststroke SHRsp receiving long-term captopril treatment. The observation that spironolactone treatment couldn't duplicate the effects of captopril suggests that aldosterone may facilitate stroke through nongenomic receptor mechanisms.

Antihyperglycemic sesquiterpenes from Psacalium decompositum.

Psacalium decompositum was investigated for antihyperglycemic compounds using diabetic ob/ob mice as a model for type 2 diabetes. In vivo bioassay-guided fractionation of an aqueous extract from the roots of P. decompositum led to the isolation of two new eremophilanolides, 3-hydroxycacalolide (1a) and epi-3-hydroxycacalolide (1b). A 1:1 mixture of 1a/1b exhibited antihyperglycemic activity when tested at 1.09 mmol/kg in ob/ob mice. The known furanoeremophilanes, cacalone (2a) and epicacalone (2b), were also isolated from the aqueous extract and were inactive. The known furanoeremophilane, cacalol (3), was isolated from a CH2Cl2 extract of P. decompositum roots and possessed antihyperglycemic activity. The relative stereochemistry in 1a and 1b was assigned 3R,5S and 3S,5S, respectively, based on ROESY data, 3J H-H values, and molecular mechanics calculations. Complete 13C and 1H NMR chemical shifts were assigned for 1a, 1b, 2a, 2b, and 3, and several revisions in 13C NMR assignments for 2a and 3 were made. Results from the conformational analysis of 1a, 1b, 2a, and 2b indicate that each compound exists in one major conformation in solution with H3-12 in a pseudoaxial position.

Antipsychotic profile of alstonine: ethnopharmacology of a traditional Nigerian botanical remedy.

Although recently developed drugs have brought significant improvement, the treatment of psychotic disorders still presents serious drawbacks. Since inherent complexity and lack of satisfactory understanding of the underlying pathophysiology impose limits for rational drug design, resourceful approaches in the search for antipsychotics are pertinent. This paper reports pharmacological properties of alstonine, a heteroyohimbine type alkaloid, which exhibited an antipsychotic-like profile, inhibiting amphetamine-induced lethality, apomorphine-induced stereotypy and potentiating barbiturate-induced sleeping time. Atypical features of alstonine were the prevention of haloperidol-induced catalepsy and lack of direct interaction with D1, D2 and 5-HT2A receptors, classically linked to antipsychotic mechanism of action.

Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) protein.

The steroidogenic acute regulatory (StAR) protein, which mediates cholesterol delivery to the inner mitochondrial membrane and the P450scc enzyme, has been shown to require a mitochondrial electrochemical gradient for its activity in vitro. To characterize the role of this gradient in cholesterol transfer, investigations were conducted in whole cells, utilizing the protonophore carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) and the potassium ionophore valinomycin. These reagents, respectively, dissipate the mitochondrial electrochemical gradient and inner mitochondrial membrane potential. Both MA-10 Leydig tumor cell steroidogenesis and mitochondrial import of StAR were inhibited by m-CCCP or valinomycin at concentrations which had only minimal effects on P450scc activity. m-CCCP also inhibited import and processing of both StAR and the truncated StAR mutants, N-19 and C-28, in transfected COS-1 cells. Steroidogenesis induced by StAR and N-47, an active N-terminally truncated StAR mutant, was reduced in transfected COS-1 cells when treated with m-CCCP. This study shows that StAR action requires a membrane potential, which may reflect a functional requirement for import of StAR into the mitochondria, or more likely, an unidentified factor which is sensitive to ionophore treatment. Furthermore, the ability of N-47 to stimulate steroidogenesis in nonsteroidogenic HepG2 liver tumor cells, suggests that the mechanism by which StAR acts may be common to many cell types.

Antipsychotic profile of alstonine: ethnopharmacology of a traditional Nigerian botanical remedy

Although recently developed drugs have brought significant improvement, the treatment of psychotic disorders still presents serious drawbacks. Since inherent complexity and lack of satisfactory understanding of the underlying pathophysiology impose limits for rational drug design, resourceful approaches in the search for antipsychotics are pertinent. This paper reports pharmacological properties of alstonine, a heteroyohimbine type alkaloid, Which exbitited an antipsychotic-like profile, inhibiting amphetamine-induced lethaly, apomorphine-induced steotypy and potentiating barbiturate-induced slleping time. Atypical features of alstonine were the prevention of haloperidol-induced catalepsy and lack of direct interaction with D1, D2 and 5-HT2A receptors, classically linked to antipsychotic mechanism of action.

Generation of retroviral vector for clinical studies using transient transfection.

Transient transfection of 293T cells was utilized to produce high-titer murine recombinant retroviral vectors for clinical studies. This system was initially optimized by gene transfer using different retroviral envelope proteins into activated human CD4+ T lymphocytes in vitro. Higher titer and infectivity were obtained than with stable murine producer lines; titers of 0.3-1 x 10(7) infectious units per milliliter for vectors encoding the green fluorescent protein (GFP) were achieved. Virions pseudotyped with envelope proteins from gibbon ape leukemia virus or amphotropic murine leukemia virus resulted in gene transfer of > or = 50% in CD4+ human T lymphocytes with this marker. Gene transfer of Rev M10 with this vector conferred resistance to HIV infection compared with negative controls in the absence of drug selection. Thus, the efficiency of transduction achieved under these conditions obviated the need to include selection to detect biologic effects in T cells. Finally, a protocol for the production of large-scale supernatants using transient transfection was optimized up to titers of 1.9 x 10(7) IU/ml. These packaging cells can be used to generate high-titer virus in sufficient quantities for clinical studies and will facilitate the rapid, cost-effective generation of improved retroviral, lentiviral, or other viral vectors for human gene therapy.

Novel terpenoid-type quinones isolated from Pycnanthus angolensis of potential utility in the treatment of type 2 diabetes.

Using an ethnomedical-based drug discovery program, two previously unknown compounds (SP-18904 and SP-18905) from Pycnanthus angolensis were isolated that lower glucose concentrations in mouse models of type 2 diabetes. SP-18904 and SP-18905 are terpenoid-type quinones that significantly lowered plasma glucose concentration (p <.05) when given orally to either ob/ob or db/db mice, both of which are hyperglycemic and hyperinsulinemic. The antihyperglycemic actions of SP-18904 and SP-18905 were associated with significant decreases in plasma insulin concentrations (p <.05), suggesting that both compounds lowered glucose by enhancing insulin-mediated glucose uptake. This was supported by the insulin suppression test in ob/ob mice. Studies in hyperglycemic, insulin-deficient mice and in vitro experiments on 3T3-L1 adipocytes further supported this conclusion. As such, these two terpenoid-type quinones represent a new class of compounds of potential use in the treatment of type 2 diabetes.

Stroke-prone spontaneously hypertensive rats lose their ability to auto-regulate cerebral blood flow prior to stroke.

OBJECTIVES: We hypothesized that the loss of cerebral blood flow (CBF) auto-regulation under hypertensive conditions could promote cerebrovascular over-perfusion and haemorrhage formation. The possibility that CBF auto-regulation becomes defective prior to haemorrhagic stroke development was assessed in Wistar- Kyoto stroke-prone spontaneously hypertensive rats (SHRsp) and related to the myogenic responsiveness of the cerebrovasculature to pressure. METHODS: Laser Doppler techniques were used to measure relative CBF in relation to mean arterial pressure (MAP 130-260 mmHg) within the perfusion domains of the middle (MCA) and posterior (PCA) cerebral arteries. The ability of isolated MCAs and PCAs to constrict to a 120 mmHg pressure step (pressure-dependent constriction) was measured using a pressure myograph. RESULTS: Two weeks prior to stroke, 10-week-old pre-stroke SHRsp exhibited near-constant CBF regulation to a 200 mmHg MAP. Thirteen-week-old pre-stroke SHRsp and age-matched post-stroke SHRsp lost their ability to auto-regulate CBF in the MCA and PCA perfusion domains. CBF increased at a high rate and in a linear manner with MAP. A distinct upper limit to CBF auto-regulation was absent. Pressure-dependent constriction was attenuated prior to stroke, and lost after stroke in isolated MCAs, but not the PCAs, of SHRsp. CONCLUSIONS: The loss of CBF auto-regulation prior to stroke in SHRsp could enhance cerebral perfusion and facilitate the initiation of haemorrhage. Such dysfunction after stroke could produce secondary haemorrhages. Defects in pressure-dependent constriction cannot fully account for the pattern of CBF auto-regulation loss observed in post-stroke SHRsp.