Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cerevisiae

Expression of both bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) were examined in Saccharomyces cerevisiae. Three ADX and two ADR expression plasmids were constructed by inserting each of the corresponding cDNA fragments between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5N. Plasmids pAX and pMX contained the coding region for the precursor and mature ADX, respectively, while pCMX carried the mature ADX preceded by the mitochondrial signal of yeast cytochrome c oxidase subunit IV (COX IV). Similarly, pMR and pCMR coded for mature ADR without and with the mitochondrial signal of yeast COX IV, respectively. Transformed S. cerevisiae AH22[rho 0]/pAX cells produced the ADX precursor, while AH22[rho 0]/pMX and AH22[rho 0]/pCMX cells produced mature ADX (mat-ADX) and modified ADX (mat-COX/ADX), respectively. Mat-ADX and mat-COX/ADX were found mainly in the cytosolic and mitochondrial fractions, respectively, and showed cytochrome c reductase activity. AH22[rho+]/pMR and AH22[rho+]/pCMR cells produced mature ADR (mat-ADR) and modified ADR (mat-COX/ADR), respectively. Mat-ADR lacking the mitochondrial signal was found in the cytosolic fraction and exhibited cytochrome c reductase activity, while mat-COX/ADR was localized in the mitochondrial fraction, but showed no reductase activity. In an in vitro reconstituted system consisting of both mat-COX/ADX- and mat-ADR-containing fractions, bovine P450scc converted cholesterol into pregnenolone. Thus mat-COX/ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450scc.     

Organella-targeted expression of rat liver cytochrome P450c27 in yeast. Genetically engineered alteration of mitochondrial P450 into a microsomal form creates a novel functional electron transport chain.

A modified rat cytochrome P450c27, whose mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of bovine cytochrome P450c17, was expressed in yeast. The modified P450c27 hemoprotein was correctly localized on yeast microsomes and exhibited the P450c27-dependent monooxygenase activity by addition of bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR). Considering the previous observation that P450c27 with its own mitochondrial targeting signal was imported into yeast mitochondria (Akiyoshi-Shibata, M., Usui, E., Sakaki, T., Yabusaki, Y., Noshiro, M., Okuda, K., and Ohkawa, H. (1991) FEBS Lett. 280, 367-370), it is now suggested that the destination of P450c27 to either mitochondria or microsomes in yeast depends solely on the amino-terminal targeting signal. In addition, the modified P450c27 was simultaneously expressed in yeast with mature forms of bovine ADX and ADR. The recombinant yeast produced the P450 on the microsomes and mature forms of ADX and ADR in the cytoplasm, and showed the monooxygenase activity. Accordingly, a novel type of functional electron transport chain has been established between the cytoplasm and the microsomes in yeast.     

A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae

The amount of glucose-repressible alcohol dehydrogenase is regulated by the amount of its functional messenger RNA. ADHII² protein was detected by a radioimmune assay and differentiated from ADHI, the classical ADH isozyme, by limited proteolysis with Staphylococcus aureus protease. When yeast containing the wild-type alleles for ADR2 (the ADH II structural locus) and for ADR1 (its positive regulatory gene) were pulse-labeled with [³⁵S]methionine during derepression, radioactive label accumulated in the antibody-precipitated ADHII coterminously with the appearance of ADHII activity. The kinetics of functional ADHII mRNA appearance during derepression in this strain were shown to be the same as those for ADHII protein synthesis in vivo when RNA, extracted from derepressed cells, was translated in a wheat germ cell-free translation system.The role of the positive regulatory gene, ADR1, in ADHII expression was analyzed using two strains mutated at that locus. Yeast containing the adr1-1 allele are incapable of derepressing ADHII activity. When this strain was pulselabeled with [³⁵S]methionine during derepression, approximately one-tenth to one-twentieth the level of ADHII protein synthesis was detected as in the wild-type strain. When RNA was extracted during derepression from cells containing the udr1-1 allele and translated in a wheat germ cell-free system, little functional ADHII mRNA was found to be present.The role of the ADR1 gene was further analyzed using a strain containing the ADR1-5^c allele, which allows constitutive synthesis of ADHII activity. In this strain during glucose repression. ADHII protein synthesis and amount of functional mRNA were at levels comparable to those found for the wild-type strain after complete derepression. Similar kinetics of ADHII protein synthesis and of mRNA accumulation during derepression were observed in the strain carrying the ADR1-5^c allele when compared to that carrying the ADR1 allele, but the absolute amounts were greater by three- to fourfold in cells containing the ADR1-5^c allele. These results indicate that the ADR1 gene acts to increase the level of functional ADHII mRNA during derepression.     

Existence of a bovine LINE repetitive insert that appears in the cDNA of bovine protein BCNT in ruminant,but not in human,genomes

A novel protein, BCNT, originally isolated from bovine brain and named after Bucentaur, contains an internal portion that is translated from part of bovine LINE repetitive sequence (Bov-B LINE). Human cDNA highly homologous to the bovine bcnt (bbcnt) cDNA has been isolated but does not contain a sequence similar to the Bov-B LINE insert (Nobukuni, T., Kobayashi, M., Omori, A., Ichinose, S., Iwanaga, T., Takahashi, I., Hashimoto, K., Hattori, S., Kaibuchi, K., Miyata, Y., Masui, T., Iwashita, S., 1997. An Alu-linked repetitive sequence corresponding to 280 amino acids is expressed in a novel bovine protein, but not in its human homologue. J. Biol. Chem. 272, 2801–2807). In this study, we conducted a polymerase chain reaction analysis to investigate whether such a Bov-B LINE insert is present in bcnt orthologs in other animals and in the genomic sequence of the human BCNT (hBCNT) gene. The results indicate that the Bov-B LINE insert is present in the genomic sequences of bcnt orthologs from sheep, goats, axis deer, and mouse deer (chevrotin), that is in Ruminantia, but not in pigs or human. Analysis of the bbcnt genomic sequence around the Bov-B LINE insert revealed a large part of the inserted Bov-B LINE sequence to be included in an exon; this is followed by a 54-nucleotide sequence that is highly homologous to Bov-B LINE in the 3′-side intron. The hBCNT gene was isolated and found to consist of seven exons and six introns, among which the intron corresponding to the Bov-B LINE insertion site in the bbcnt genome is 16.5 kb in length with no sequence similar to Bov-B LINE. Based on these results, it seems likely that the Bov-B LINE insert is derived from a long Bov-B LINE repetitive sequence transposed to an ancestral bcnt gene in Ruminantia and reformed as a new exon through new splicing sites in the transposed sequence.     

Mixed-function oxidase enzymes in adriamycin-sensitive and resistant sublines of P-388 leukemia

Levels of mixed-function oxidase (MFO) enzymes were measured in adriamycin(ADR)-sensitive murine leukemia P-388 and its ADR-resistant subline P-388/ADR. The subcellular fractions of the resistant cells showed decreased contents of MFO components, cytochrome P-450 and cytochrome b₅, in comparison with the identically prepared fractions of the parental tumor. Similarly, the levels of 7-ethoxycoumarin O-deethylase and the rate of ascorbate induced lipid peroxidation in vitro showed lower values in resistant tumor cells than those of P-388 tumor cells. The observed differences in the two tumor cell types were found to be considerably enhanced if the tumor cells were exposed in vitro to ADR before fractionation. The magnitude of induction of the MFO enzymes was significantly greater in the ADR exposed P-388 cells. The corresponding inducibility was suppressed in the drug exposed resistant tumor cells.     

Dissociation of hyperreninemia and renal prostaglandin synthesis in the adrenalectomized rat

The aim of this study was to determine whether hyperreninemia in the adrenalectomized (ADX) rat is dependent on renal prostaglandin synthesis, as has been suggested for two other hyperreninemic conditions, Bartter's syndrome and chronic liver disease.Plasma renin concentration (PRC) in anesthetized, ADX rats was significantly increased (Δ +480%; p < 0.001) compared to sham-operated controls. , indomethacin (10 mg/kg i.v.) significantly reduced PRC of anesthetized, ADX rats after both 45 min (Δ −34%; p < 0.05) and 90 min (Δ −47%; p < 0.05). renin release from renal cortical slices of ADX rats was also significantly greater (Δ +130%; p < 0.05) than from sham-operated control cortical slices. Renin release from cortical slices of ADX rats given dexamethasone (10 μg/kg/day) for 4 days prior to sacrifice did not differ from sham-operated control values.Prostaglandin E₂ (PGE₂) release from cortical slices of ADX rats did not differ significantly from controls. However, PGE₂ synthesis in glomeruli microdissected from ADX rats was significantly increased (Δ +110%; p < 0.001) compared to controls. PGE₂ synthesis in glomeruli of dexamethasone-treated ADX rats remained significantly elevated compared to controls. Ibuprofen (10⁻⁶ M) decreased PGE₂ synthesis in cortical slices by 80%. However, prostaglandin synthesis inhibition had no effect on renin release from either ADX or control renal cortical slices.These results suggest that despite increased glomerular synthesis, prostaglandins do not directly influence renin release in the ADX rat.     

The inhibitory effect of kokusaginine on the growth of human breast cancer cells and MDR-resistant cells is mediated by the inhibition of tubulin assembly

The emergence of multidrug resistance (MDR) is a significant challenge in breast carcinoma chemotherapy. Kokusaginine isolated from Dictamnus dasycarpus Turcz. has been reported to show cytotoxicity in several human cancer cell lines including breast cancer cells MCF-7. In this study, kokusaginine showed the potent inhibitory effect on MCF-7 multidrug resistant subline MCF-7/ADR and MDA-MB-231 multidrug resistant subline MDA-MB-231/ADR. Kokusaginine markedly induced apoptosis in a concentration-dependent manner in MCF-7/ADR cells. Furthermore, kokusaginine reduced P-gp mRNA and protein levels, and suppressed P-gp function especially in MCF-7/ADR cells. In addition, kokusaginine showed to inhibit tubulin assembly and the binding of colchicine to tubulin by binding directly to tubulin and affects tubulin formation in vitro. Taken together, these results support the potential therapeutic value of kokusaginine as an anti-MDR agent in chemotherapy for breast carcinoma.     

Adrenalectomy Reduces Adiposity by Decreasing Feed Efficiency,Not Direct Effects on White Adipose Tissue

EDENS, N. K., A. MOSHIRFAR, G. M. POTTER, S. K. FRIED, AND T. W. CASTONGUAY. Adrenalectomy reduces adiposity by decreasing feed efficiency, not direct effects on white adipose tissue. Obes Res. Objective: This study was conducted to establish the effects of adrenalectomy (ADX) on adipose tissue metabolism in male Sprague—Dawley rats fed a standard chow diet. Research Methods and Procedures: The effects of adrenalectomy on adipose cell size, lipoprotein lipase activity, and basal and insulin-stimulated glucose conversion to lipid and lipolysis were measured. Results: ADX decreased body weight gain during the postoperative period in the absence of changes in food intake; feed efficiency was decreased significantly. ADX decreased adipocyte size by 30%. ADX increased adipocyte response to the effect of submaximal concentrations of insulin on lipid synthesis and lipolysis. ADX decreased maximally insulin-stimulated lipid synthesis, but this effect was accounted for by decreased adipocyte size. In contrast, ADX had no effect on maximally insulin-inhibited lipolysis. ADX did not affect heparin-releasable LPL. The small effect of ADX on residual extractable adipose tissue LPL activity was accounted for by decreased fat cell size. Discussion: ADX decreased adiposity in the absence of changes in food intake, lipoprotein lipase activity, and adipocyte lipid metabolism. The effect is best attributed to decreased feed efficiency.     

Long Noncoding RNA MRUL Promotes ABCB1 Expression in Multidrug-Resistant Gastric Cancer Cell Sublines

Multidrug resistance (MDR) is the most common cause of chemotherapy failure in gastric cancer (GC) treatment; however, the underlying molecular mechanisms remain elusive. Long noncoding RNAs (lncRNAs) can be involved in carcinogenesis, but the effects of lncRNAs on MDR are poorly understood. We show here that the lncRNA MRUL (MDR-related and upregulated lncRNA), located 400 kb downstream of ABCB1 (ATP-binding cassette, subfamily B, member 1), was significantly upregulated in two multidrug-resistant GC cell sublines, SGC7901/ADR and SGC7901/VCR. Furthermore, the relative expression levels of MRUL in GC tissues were negatively correlated with in vitro growth inhibition rates of GC specimens treated with chemotherapeutic drugs and indicated a poor prognosis for GC patients. MRUL knockdown in SGC7901/ADR and SGC7901/VCR cells led to increased rates of apoptosis, increased accumulation, and reduced doxorubicin (Adriamycin [ADR]) release in the presence of ADR or vincristine. Moreover, MRUL depletion reduced ABCB1 mRNA levels in a dose- and time-dependent manner. Heterologous luciferase reporter assays demonstrated that MRUL might positively affect ABCB1 expression in an orientation- and position-independent manner. Our findings indicate that MRUL promotes ABCB1 expression and is a potential target to reverse the MDR phenotype of GC MDR cell sublines.     

Potential Therapeutic Effect of Natural Killer Cells on Doxorubicin-Resistant Breast Cancer Cells In Vitro

Objective

The aim of this study was to explore the therapeutic effect of natural killer (NK) cells on human doxorubicin-sensitive and resistant breast adenocarcinoma.

Methods

Human doxorubicin-sensitive and resistant breast cancer cell lines (MCF-7 and MCF-7/ADR) were tagged with renilla luciferase (Rluc) (MCF-7/RC and MCF-7/ADR/RC). NK cells were tagged with enhanced firefly luciferase (effluc) using a recombinant retrovirus transfection (NKF). Expression of Rluc, effluc, and NK cell surface markers CD16, CD56 as well as death receptors, DR4 and DR5, were assessed by using flow cytometry. In vitro cytotoxic effect of NK to MCF-7 and MCF-7/ADR was measured and in vivo bioluminescence imaging was also performed to visualize MCF-7/RC, MCF-7/ADR, and NKF in an animal model.

Results

NK92-MI, MCF-7, and MCF-7/ADR cells were successfully labeled with Rluc or effluc. Both the target breast cancer cells (with Rluc) and therapeutic NK cells (with effluc) were noninvasively visualized in nude mice. Doxorubicin-resistant breast cancer cells (MCF-7/ADR) presented a higher expression of DR5 and were more sensitive to NK cells compared with doxorubicin-sensitive breast cancer cells (MCF-7).

Conclusion

The results of present study suggest that NK cell therapy has a therapeutic effect on doxorubicin-sensitive and resistant breast cancer cells.     

The β3-adrenergic receptor in the obesity and diabetes prone rhesus monkey is very similar to human and contains arginine at codon 64

The β₃-adrenergic receptor (ADRβ₃) is a seven-membrane spanning, G-protein linked receptor expressed in brown adipose tissue in rodents, and visceral adipose tissue in humans. Stimulation of the receptor by norepinephrine leads to lipolysis and thermogenesis. In rodent models of obesity and diabetes, administration of β₃-agonists results in weight loss and improved glucose tolerance. Studies indicate that the pharmacological properties of the ADRβ₃ differ markedly between rodents and humans, making generalizations of rodent studies to humans difficult. We hypothesized that the obesity and diabetes prone rhesus monkey (Macaca mulatta) would provide an excellent animal model to study the role of the ADRβ₃ in the development of obesity and diabetes as well as for assessment of the therapeutic efficacy of β₃-agonists. We sequenced the entire coding region of the rhesus ADRβ₃ gene. Like humans, the rhesus ADRβ₃ has two exons. There is 89% amino acid (aa) identity between human and rhesus compared to 82% aa identity between human and mouse. A single base deletion results in divergence of the intracellular carboxy terminus accounting for 26 of the 45 aa changes and 10 additional aa. Of the 15 rhesus monkeys studied, all were homozygous for Arg⁶⁴. In humans, Arg⁶⁴ (rather than Trp) is associated with increased body mass index, insulin resistance, and an earlier onset of type II diabetes mellitus. We conclude that the rhesus ADRβ₃ is more similar to the human ADRβ₃ than to the rodent ADRβ₃ suggesting that this primate model may be more appropriate for physiologic and therapeutic studies of the ADRβ₃ axis, and that Arg⁶⁴ may influence susceptibility in this species to obesity, insulin resistance, and type II diabetes.     

[125I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes

[¹²⁵I]LSD (labeled at the 2 position) has been introduced as the first ¹²⁵I-labeled ligand for serotonin 5-HT₂ (S2) receptors. In the present study we examined the binding of [¹²⁵I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [¹²⁵I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal K_d = 1.1 nM, B_max = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC₅₀ values for a variety of compounds show a clear 5-HT₂ (S2) serotonergic pattern at this [¹²⁵I]LSD site. Blockage of this primary 5-HT₂ (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [¹²⁵I]LSD binding site with a K_d = 9.1 nM, B_max = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [¹²⁵I]LSD is a sensitive and selective label for 5-HT₂ (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [¹²⁵I]LSD binding site with mianserin allows the high sensitivity of [¹²⁵I]LSD to be applied to D2 dopamine receptor studies as well.     

A Link between Aurora Kinase and Clp1/Cdc14 Regulation Uncovered by the Identification of a Fission Yeast Borealin-Like Protein

The chromosomal passenger complex (CPC) regulates various events in cell division. This complex is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin. Together, these four subunits interdependently regulate CPC function, and they are highly conserved among eukaryotes. However, a Borealin homologue has never been characterized in the fission yeast, Schizosaccharomyces pombe. Here, we isolate a previously uncharacterized S. pombe protein through association with the Cdc14 phosphatase homologue, Clp1/Flp1, and identify it as a Borealin-like member of the CPC. Nbl1 (novel Borealin-like 1) physically associates with known CPC components, affects the kinase activity and stability of the S. pombe Aurora B homologue, Ark1, colocalizes with known CPC subunits during mitosis, and shows sequence similarity to human Borealin. Further analysis of the Clp1–Nbl1 interaction indicates that Clp1 requires CPC activity for proper accumulation at the contractile ring (CR). Consistent with this, we describe negative genetic interactions between mutant alleles of CPC and CR components. Thus, this study characterizes a fission yeast Borealin homologue and reveals a previously unrecognized connection between the CPC and the process of cytokinesis in S. pombe.     

Hepatic Gluconeogenesis and the Activity of PDH in Individual Tissues of GTG-Obese Mice Following Adrenalectomy

Adrenalectomy (ADX) lowers circulating glucose levels in animal models of non-insulin dependent diabetes (NIDDM) and obesity. To investigate the role of hepatic glucose production (HGP) and tissue glucose oxidation in the improvement in glucose tolerance, hepatocyte gluconeogenesis and the activity of pyruvate dehydrogenase (PDH) were examined in different tissues of gold thioglucose (GTG) obese mice 2 weeks after ADX or sham ADX. GTG-obese mice which had undergone ADX weighed significantly less than their adrenal intact counterparts (GTG ADX: 37.5 ± 0.7g; GTG: 44.1 ± 0.4g; p<0.05), and demonstrated lower serum glucose (GTG ADX: 22.5 ± 1.6 mmol/L; GTG: 29.4 ± 1.9 mmol/L; p<0.05) and serum insulin levels (GTG ADX: 76 ± 10μ.U/mL; GTG: 470 ± 63μU/mL; p<0.05). Lactate conversion to glucose by hepatocytes isolated from ADX GTG mice was significantly reduced compared with that of hepatocytes from GTG mice (GTG ADX: 125 ± 10 nmol glucose/10⁶ cells; GTG: 403 ± 65 nmol glucose/10⁶ cells; p<0.05). ADX also significantly reduced both the glycogen (GTG ADX: 165 ± 27 μmol/liver; GTG: 614 ± 60 pmol/Iiver; p<0.05) and fatty acid content (GTG ADX: 101 ± 9 mg fatty acid/g liver; GTG: 404 ± 40 mg fatty acid/g liver; p<0.05) of the liver of GTG-obese mice. ADX of GTG-obese mice reduced PDH activity by varying degrees in all tissues, except quadriceps muscle. These observations are consistent with an ADX induced decrease in hepatic lipid stores removing fatty acid-induced increases in gluconeogenesis and increased peripheral availability of fatty acids inhibiting PDH activity via the glucose/fatty acid cycle. It is also evident that the improvement in glucose tolerance which accompanies ADX of GTG-obese mice is not due to increased PDH activity resulting in enhanced peripheral glucose oxidation. Instead, it is more likely that reduced blood glucose levels after ADX of GTG-obese mice are the result of decreased gluconeogenesis in the liver.     

3D Structure Prediction of Human β1-Adrenergic Receptor via Threading-Based Homology Modeling for Implications in Structure-Based Drug Designing

Dilated cardiomyopathy is a disease of left ventricular dysfunction accompanied by impairment of the β₁-adrenergic receptor (β₁-AR) signal cascade. The disturbed β₁-AR function may be based on an elevated sympathetic tone observed in patients with heart failure. Prolonged adrenergic stimulation may induce metabolic and electrophysiological disturbances in the myocardium, resulting in tachyarrhythmia that leads to the development of heart failure in human and sudden death. Hence, β₁-AR is considered as a promising drug target but attempts to develop effective and specific drug against this tempting pharmaceutical target is slowed down due to the lack of 3D structure of Homo sapiens β₁-AR (hsβADR1). This study encompasses elucidation of 3D structural and physicochemical properties of hsβADR1 via threading-based homology modeling. Furthermore, the docking performance of several docking programs including Surflex-Dock, FRED, and GOLD were validated by re-docking and cross-docking experiments. GOLD and Surflex-Dock performed best in re-docking and cross docking experiments, respectively. Consequently, Surflex-Dock was used to predict the binding modes of four hsβADR1 agonists. This study provides clear understanding of hsβADR1 structure and its binding mechanism, thus help in providing the remedial solutions of cardiovascular, effective treatment of asthma and other diseases caused by malfunctioning of the target protein.