Milligram Of Protein Research Articles

Protective effects of melatonin and N‐acetylserotonin on aflatoxin B1‐induced lipid peroxidation in rats

Aflatoxin B1 (AFB1) is a potent hepatotoxic and hepatocarcinogenic mycotoxin. Reactive oxygen species are considered to participate in the main mechanism of aflatoxin toxicity. Melatonin (Mel) is a hormone which has antioxidative activities. N-acetylserotonin (NAc-5HT) is an immediate precursor of Mel. Melatonin is documented to be completely safe in humans and animals. The aim of our study was to examine the potential protective effects of Mel or NAc-5HT against lipid peroxidation (LPO), caused by AFB1 in male Wistar rats. Mel and NAc-5HT were intraperitoneally (i.p.) injected for 3 weeks in late afternoon (16:00-18:00) injections (20 mg kg(-1) BW/daily). AFB1 (50 microg kg(-1) BW/daily) was administered i.p. 6 h prior to indoleamine injections. Concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA), as an index of LPO, were measured in liver, brain, lung, testis and kidney homogenates. The level of LPO in tissue homogenates was expressed as the amount of MDA + 4-HDA (nmol) per milligram of protein. AFB1 increased LPO in the liver, lung, brain and testis, but not the kidney. The increase of LPO caused by AFB1 injections was completely prevented by either Mel or NAc-5HT in all the tissues examined. Melatonin can be considered as a protective pharmacological agent in intoxication with AFB1 and the protective effect of NAc-5HT against aflatoxin-induced LPO broadens the knowledge about its antioxidative properties.

Expression and Function of the Calcium-Sensing Receptor in Juxtaglomerular Cells

Calcium-sensing receptors sense and translate micromolar changes of extracellular calcium into changes in intracellular calcium. Renin, a component of the renin-angiotensin system, is synthesized by, stored in, and released from the juxtaglomerular cells through a cAMP-dependent pathway. Increased intracellular calcium inhibits the adenylyl cyclase isoform type V, cAMP formation, and renin release from juxtaglomerular cells. We hypothesized that calcium-sensing receptors are expressed in juxtaglomerular cells and mediate changes in intracellular calcium and renin release. To test this we used primary cultures of isolated mouse juxtaglomerular cells in which we ran RT-PCR, Western blots, and immunofluorescence. RT-PCR showed a positive band at the expected 151 bp consistent with calcium-sensing receptor. Western blots showed a 130- to 150-kDa band confirming the calcium-sensing receptor in juxtaglomerular cells. Immunofluorescence and confocal microscopy using 2 different antibodies against the calcium-sensing receptor in juxtaglomerular cells showed positive fluorescence in the juxtaglomerular cells, which also had positive labeling for renin. To test whether calcium-sensing receptors regulate renin release, juxtaglomerular cells were incubated with a calcium-sensing receptor agonist, the calcimimetic cinacalcet-HCl, at concentrations of 50 and 1000 nmol/L in 0.25 mmol/L of calcium medium. Cinacalcet-HCl decreased juxtaglomerular cell cAMP formation to 47.3+/-6.8% and 44.2+/-9.7% of basal, respectively (P<0.001), and decreased renin release from 541.9+/-86.2 to 364.6+/-64.1 (P<0.05) and 279.6+/-56.9 (P<0.005) ng of angiotensin I per milliliter per hour per milligram of protein, respectively. We conclude that juxtaglomerular cells express the calcium-sensing receptor and that their activation leads to inhibition of adenylyl cyclase-V activity, decreasing cAMP formation and suppressing renin release.

Recombinant Sea Urchin Flagellar Adenylate Kinase

Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 mumol ATP formed per minute per milligram of protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 microM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.

High‐density transient gene expression in suspension‐adapted 293 EBNA1 cells

Large-scale transient gene expression (TGE) in mammalian cells is an attractive method to rapidly produce recombinant proteins for pre-clinical studies, with some processes reported to reach 100 L. However, the yield remains low, hardly over 20 mg protein/L, mainly because the current TGEs have been performed at low cell density (approximately 5 x 10(5) cells/mL). In this study, the strategy to improve TGE focuses on facilitating transfection at high cell density. A high-density perfusion culture of 293 EBNA1 cells was established in 2-L bioreactor using Freestyle 293 expression medium (Invitrogen, Singapore) to grow the cells for transfection. Transfection was then carried out at 1 x 10(7) cells/mL using polyethylenimine (PEI) as DNA carrier, at the optimized conditions of 6 microg DNA/10(7) cells and 1:3 DNA to PEI mass ratio. During the post-transfection phase, 80.8 mg/L of the model protein, EPO was obtained at day 5.5 post-transfection (130 mg total EPO production) using a fed-batch culture mode. In comparison, perfusion cultures using an enriched SFM II medium resulted in a longer post-transfection production phase (8 days), and 227 mg of EPO was produced in 10.7 L medium, showing that high-density TGE enables the production of several hundreds of milligrams of protein in a 2 L bioreactor. In addition, a protocol for economical plasmid preparation based on anion exchange was also established to satisfy TGE's demand in terms of quality and quantity. To the best of our knowledge, this is the first report of transient transfections at a high cell density of up to 1 x 10(7) cells/mL.

Expression of a Rice Chlorophyll a/b Binding Protein Promoter in Sweetpotato

Leaves are usually the target tissue for expressing transgenes conferring resistances to herbicides, pests, and diseases. To achieve leaf-specific expression, a light-harvest chlorophyll a/b binding protein (CAB) of photosystem-II (CAB2) promoter (CAB2-p) from rice (Oryza sativa L.) and the cauliflower mosaic virus 35S promoter were fused to the β-glucuronidase (GUS) reporter and subsequently evaluated in transgenic sweetpotato [Ipomoea batatas L. (Lam.)]. The 35S promoter-directed GUS activities varied from 46.0 to 61.2 nmol 4-methyl-umbelliferyl-β-D-glucuronide (4-MU) per minute per milligram of protein in leaf, stem, primary, and storage roots. In contrast, the CAB2-p directed an uneven distribution of GUS activities (4-MU at 1.1 to 12.6 nmol·min−1·mg−1 protein); GUS activity in mature leaves was ≈12-fold as high as that in storage roots. In addition, GUS assay in leaf tissues revealed that CAB2-p enabled a developmentally controlled and light-regulated GUS expression. These results indicate that the rice CAB2-p could be used to drive leaf-specific expression of linked genes in sweetpotato.

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.

Angiotensin-Converting Enzyme I/D and α-Adducin Gly460Trp Polymorphisms

The angiotensin-converting enzyme (ACE) I/D and the alpha-adducin (ADD1) Gly460Trp polymorphisms are associated with cardiovascular risk factors. In a prospective population study and in cell models, we investigated the combined effects of these 2 polymorphisms. We randomly recruited 1287 white subjects (women: 50.0%; mean age: 55.9 years). We obtained outcomes from registries and repeat examinations (median 3). Over 9.0 years (median), 178 fatal or nonfatal cardiovascular events occurred. In ADD1 Trp allele carriers, the multivariate-adjusted hazard ratios associated with ACE DD versus I were 1.72 (P=0.007) for total mortality, 2.35 (P=0.02) for cardiovascular mortality, 2.02 (P=0.005) for all cardiovascular events, and 2.59 (P=0.03) for heart failure. In contrast, these hazard ratios did not reach significance in ADD1 GlyGly homozygotes (0.08<or=P<or=0.90). The positive predictive value and attributable risk associated with ACE DD homozygosity combined with mutated ADD1 were 36.2% and 10.3%, respectively. To clarify our epidemiological observations, we investigated the effects of mutated human ADD1 on the membrane-bound ACE activity in fibroblasts from 51 volunteers and in transfected human embryonic kidney cells (31 experiments). In fibroblasts (5.10 versus 3.63 nanomoles of generated hippuric acid per milligram of protein per minute; P=0.0021) and human embryonic kidney cells (1.086 versus 0.081 nmol/mg per minute; P=0.017), the membrane-bound ACE activity increased in the presence but not absence of the ADD1 Trp allele. In conclusion, the combination of ACE DD homozygosity and mutated ADD1 worsened cardiovascular prognosis to a similar extent as classic risk factors, possibly because of increased membrane-bound ACE activity in subjects carrying the ADD1 Trp allele.

Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Melatonin and its synthesizing enzymes (arylalkylamine N‐acetyltransferase‐like and hydroxyindole‐O‐methyltransferase) in avian eggs and early embryos

The presence of melatonin and the enzymes (transcripts and activities) involved in its synthesis, i.e. arylalkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), was investigated in the eggs and early embryos of Japanese quail at Hamburger-Hamilton stages 1-10. Melatonin was present in the egg yolk (approximately 70 pg/g) and albumen (approximately 20 pg/g). The average content of melatonin was approximately 416 pg/egg. AA-NAT and HIOMT transcripts were present in the oocytes, blastoderms, and ovarian follicles. AA-NAT-like and HIOMT activities were detected in quail egg yolk. The activity of AA-NAT in yolk was comparable with that found in the pineal gland when calculated per milligram of yolk or pineal gland, but was significantly lower when re-calculated per milligram of protein in the yolk or pineal gland. AA-NAT-like activity was also identified in the ovarian follicles. Low HIOMT activity was detected in yolk, but not in the ovarian follicle. Both enzymes were essentially absent from early embryos although some residual activities, probably of yolk origin, were present in the stage 1 embryo. Melatonin and all the constituents needed for its synthesis (serotonin, AA-NAT and HIOMT activities) are contained within the avian yolk and could be used by the embryo from the very beginning of its development. The role of extrapineal melatonin in early avian development may be in protecting the embryo from the action of free radicals formed during intensive embryonic metabolism and/or it may participate (together with serotonin) in a 'diffuse neuroendocrine system' acting at early developmental stages, before differentiation of the nervous system.

Nutlin-3 Protects Kidney Cells during Cisplatin Therapy by Suppressing Bax/Bak Activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.

Matrix metalloproteinase and tissue inhibitor expression in atherosclerotic and nonatherosclerotic thoracic aortic aneurysms

The altered expression of matrix metalloproteinases and their inhibitors influences the formation of atherosclerotic abdominal aortic aneurysms. Their association with thoracic aneurysms is less clear. This study describes the expression of metalloproteinases and their inhibitors in atherosclerotic and nonatherosclerotic thoracic aneurysms, and compares these with age-matched controls. Matrix metalloproteinase-2 and 9 activity were measured by antibody capture, and tissue inhibitor-1 and 2 levels were measured by enzyme-linked immunosorbent assay in 24 patients with atherosclerotic aneurysms and in 63 patients with nonatherosclerotic aneurysms. Gene expression was assessed with reverse transcriptase polymerase chain reaction. The results were compared with 17 controls. Data are in nanograms per milligram of protein. Matrix metalloproteinase-2 activity was greater in controls than in the atherosclerotic and nonatherosclerotic groups (80 +/- 67 vs 49 +/- 50 and 35 +/- 44, P = .002). Matrix metalloproteinase-9 activity was greater in the atherosclerotic group than in the nonatherosclerotic group and controls (11.7 +/- 15.7 vs 2.5 +/- 2.2 and 1.7 +/- 1.9, P = .001). Tissue inhibitor-1 and 2 levels were greater in controls than in either aneurysm group (tissue inhibitor of metalloproteinase-1: 376 +/- 192 vs 234 +/- 233 and 174 +/- 148, P = .003; tissue inhibitor of metalloproteinase-2: 143 +/- 74 vs 14 +/- 13 and 27 +/- 43, P < .001). Atherosclerotic aneurysms expressed more matrix metalloproteinase mRNA than controls. The metalloproteinase/tissue inhibitor phenotype of atherosclerotic thoracic aneurysms is similar to that of abdominal aneurysms. The diminished expression of metalloproteinases and tissue inhibitors in nonatherosclerotic thoracic aneurysms relative to aged controls may represent a loss of smooth muscle cells.

Adenylyl Cyclase Isoform V Mediates Renin Release From Juxtaglomerular Cells

We have shown previously that decreasing intracellular calcium in the juxtaglomerular cells increases both cAMP formation and renin release. We hypothesized that this is because of an interaction between intracellular calcium and the calcium-inhibitable isoform of adenylyl cyclase, type-V. We used primary cultures of juxtaglomerular cells isolated from C-57/B6 mice at 70% to 80% confluence. Western blots were performed on isolated juxtaglomerular cells using antibodies against either of the 2 calcium inhibitable isoforms of adenylyl cyclase, types-V and -VI. Only the antibody against adenylyl cyclase-V gave us a strong band at 120 kDa as expected. Immunolabeling in juxtaglomerular cells with confocal microscopy found immunofluorescence for the adenylyl cyclase-V-specific antibody compared with either negative controls or cells stained with the adenylyl cyclase-VI antibody. Reducing isolated juxtaglomerular intracellular calcium with 100 micromol/L of the cytosolic calcium chelator BAPTA-AM stimulated both cAMP (3.49+/-0.70 to 10.09+/-0.81 pmol/mL per milligram of protein; P<0.002) and renin release (1001.8+/-81.5 to 1648.0+/-139.1 ng of angiotensin I per milliliter per hour per milligram of protein; P<0.01). The selective adenylyl cyclase-V inhibitor NKY80 completely blocked both BAPTA-AM-stimulated cAMP formation and renin release. We conclude that lowering intracellular calcium is permissive, allowing an increased activity of the calcium-inhibitable isoform adenylyl cyclase-V (but not adenylyl cyclase-VI) in the juxtaglomerular cell, producing cAMP, which stimulates renin secretion.

Decreased Intracellular Calcium Stimulates Renin Release via Calcium-Inhibitable Adenylyl Cyclase

Intracellular calcium and cAMP are the 2 second messengers that regulate renin release; cAMP stimulates renin release from juxtaglomerular (JG) cells, whereas increased intracellular calcium inhibits it. We hypothesized that decreased intracellular calcium acts by activating calcium-inhibitable isoforms of adenylyl cyclase, increasing cAMP, and stimulating renin secretion. We used a primary culture of JG cells isolated from C-57/B6 mice. Cells were plated to a density of 70% in serum-free medium and incubated for 2 hours with or without 100 micromol/L of the cytosolic calcium chelator 5'5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Intracellular cAMP content was 4.04+/-0.92 pM/mL per milligram of protein, and it increased by125+/-33% (P<0.01) with BAPTA-AM. Basal renin was 1.28+/-0.40 microg of angiotensin I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 182+/-62% (P<0.025). Western blots using an antibody that recognizes adenylyl cyclase types V and VI yielded a characteristic band of approximately 135 kDa. When primary cultures of isolated JG cells were tested for the calcium-inhibitable isoforms of adenylyl cyclase, they showed intense focal cytoplasmic staining. Cells stained for both renin and adenylyl cyclase V/VI showed colocalization in the cytoplasm, primarily on the granules. An adenylyl cyclase inhibitor (SQ 22,536) completely blocked BAPTA-AM-stimulated renin release and JG cell cAMP content. We conclude that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Thus, decreased intracellular calcium stimulates adenylyl cyclase, resulting in cAMP synthesis and, consequently, renin release.

Inflammatory Murine Skin Responses to UV-B Light Are Partially Dependent on Endothelin-1 and Mast Cells

Endothelin (ET-1) has been shown to crucially contribute to UV-induced skin responses such as tanning. To test whether ET-1 is also involved in early cutaneous reactions to UV, we assessed ET-1 skin levels in UV-irradiated mice. In correlation with the levels of UV-induced skin inflammation, ET-1 concentrations increased substantially and continually. Moreover, blocking of ET-1 receptors (ETA) resulted in significantly decreased cutaneous inflammation following UV irradiation. When we assessed skin responses to ET-1 injections, we observed prominent mast cell degranulation and mast cell-dependent inflammation. Since mast cells also critically contributed to UV-induced inflammation, we determined the ET-1-dependent inflammatory response to UV in the absence and presence of these cells. Interestingly, ETA blockade did not decrease UV-induced inflammation in mast cell-deficient mice, unless these mice had been adoptively transferred with mast cells before irradiation. This indicates that skin inflammation due to UV irradiation is caused in part by ET-1 acting on skin mast cells.

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 μCi of [ 14C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 μmol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min·mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K m of 38 ± 25 and 45 ± 15 μmol/L, whereas the K m for retigabine N-glucuronidation by human liver microsomal fractions was 145 ± 39 μmol/L. Furthermore, a V max of 1.2 ± 0.3 (nmol/[min·mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine– N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 μmol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of 14C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.

In vivo electroporation and ubiquitin promoter – a protocol for sustained gene expression in the lung

Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.

Adrenomedullin Peptide: Gene Expression of Adrenomedullin, its Receptors and Receptor Activity Modifying Proteins, and Receptor Binding in Rat Testis—Actions on Testosterone Secretion1

Adrenomedullin (ADM) has been shown to be present in the human and rat male reproductive systems. This study demonstrates the expression of ADM in the rat testis and its effect on the secretion of testosterone. Whole testicular extracts had 5.43 +/- 0.42 fmol of immunoreactive ADM per milligram of protein and 84 +/- 8 fg of ADM mRNA per picogram of Actb (beta-actin) mRNA. Immunocytochemical studies showed positive ADM immunostaining in the Leydig cells and in the Sertoli cells. Gel filtration chromatography of testicular extracts showed two peaks, with the predominant one eluting at the position of the ADM precursor. Furthermore, the testis was shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity modifying protein 1 (Ramp1), Ramp2, and Ramp3. These account for the specific binding of ADM to the testis, which was partially inhibited by human ADM (22-52) and by human calcitonin gene-related peptide (8-37), the ADM and calcitonin gene-related peptide receptor antagonists, respectively. Administration of ADM to testicular blocks in vitro resulted in a dose-dependent inhibition of hCG-stimulated release of testosterone, which was abolished by the administration of ADM (22-52). Our results suggest a paracrine effect of ADM on testicular steroidogenesis.

Overexpression, purification and preliminary X-ray analysis of pullulanase fromBacillus subtilisstrain 168

The AmyX gene encoding pullulanase from the common spore-forming bacterium Bacillus subtilis strain 168 was cloned, overexpressed in Escherichia coli, purified and crystallized. The recombinant pullulanase was purified to homogeneity using ammonium sulfate precipitation, hydrophobic chromatography and anion-exchange chromatography, resulting in a specific activity of 24.10 U per milligram of protein. SDS-PAGE analysis showed that the molecular weight of the protein is approximately 81.0 kDa, which is similar to the calculated molecular weight, 81.1 kDa, from its translated cDNA sequence. The k(cat) and K(m) of the purified enzyme with pullulan as substrate were approximately 79 s(-1) and 1.284 mg ml(-1), respectively. X-ray crystallographic analysis of the pullulanase crystal showed that the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.568, b = 127.68, c = 189.25 angstroms. The crystal contains two molecules of pullulanase in the asymmetric unit, with a solvent content of 53.15%. The crystal diffracted to 2.1 angstroms resolution at a synchrotron and is suitable for structure determination.

Expression, purification, and circular dichroism analysis of human CDK9

The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a vector at 30 °C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors.

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this alpha-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH.