Effects Of Isopropyl Research Articles

Optimization of recombinant β-NGF expression in Escherichia coli using response surface methodology

ABSTRACTHuman nerve growth factor a member of the neurotrophin family can be used to treat neurodegenerative diseases. As it has disulfide bonds in its structure, periplasmic expression of it using appropriate signal sequence is beneficial. Therefore, in this work β-nerve growth factor (β-NGF) was expressed in Escherichia coli using pET39b expression vector containing DsbA signal sequence. In an initial step, the effect of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose concentration as inducer on protein production was investigated using response surface methodology. Then the effect of different postinduction time and temperature on protein production was studied. Our results indicated that the highest β-NGF production was achieved with 1 mM IPTG and low concentrations of lactose (0–2% w/v), low cultivation temperature of 25°C and postinduction time of 2 hr. Also following β-NGF purification, bioassay test using PC12 cell line was done. The biological activity of the purified β-NGF showed a similar cell proliferation activity with the standard recombinant human β-NGF. In conclusion, the results indicated an optimized upstream process to obtain high yields of biologically active β-NGF.

Heterologous expression and purification of porcine fat mass andobesity-associated gene in Escherichia coli

A porcine fat mass and obesity-associated gene (pFTO) was cloned into the expression vector pET30a(+) and heterologously expressed in Escherichia coli. The effects of isopropyl ß-D-thiogalactopyranoside (IPTG) concentration and induction time on the expression of recombinant pFTO were assessed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the molecular mass of the recombinant pFTO protein was about 56 kDa, and the recombinant protein could be induced efficiently in E. coli BL21 by the addition of 0.75 mM IPTG for 4 h at 30 °C. The recombinant pFTO fusion protein was purified using Ni-IDA affinity chromatography with a yield of about 1.8 µg/mL protein. The protein was further confirmed by western blot analysis. An in vitro biological activity assay demonstrated that the refolded purified 2 µg/mL recombinant pFTO protein increased 3T3-L1 preadipocyte proliferation. The present work should be useful for the production of sufficiently large amounts of recombinant pFTO protein for further functional analysis.

Molecular cloning and expression of the pyranose 2-oxidase cDNA from Trametes ochracea MB49 in Escherichia coli.

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28 degrees C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg(-1)). The effect of isopropyl beta-D-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28 degrees C. The synthesis of P2O at 28 degrees C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg(-1)). At 25 degrees C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg(-1). The soluble enzyme represented 70% of the total amount of P2O.

Synthesis and calcium channel modulation effects of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-phenylpyridine-5-carboxylates possessingortho-,meta-, andpara-CH2S(O)nMe and -S(O)nMe (n = 0-2) phenyl substituents

A group of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-phenylpyridine-5-carboxylates (13–15) possessing ortho-, meta-, and para-CH2S(O)nMe and –S(O)nMe (n = 0–2) phenyl substituents were synthesized using a modified Hantzsch reaction. Calcium channel (CC) modulating activities were determined using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) in vitro assays. This class of –CH2S(O)nMe and –S(O)nMe (n = 0–2) compounds (13–15a–f) exhibited weaker CC antagonist activity on GPILSM (IC50 = > 1.1 × 10–5 to 4.1 × 10–6 M range) than the reference drug nifedipine (IC50 = 1.4 × 10–8 M). The oxidation state of the sulfur atom was a determinant of smooth muscle CC antagonist activity where the relative activity profile was generally thio (13, -CH2SMe, -SMe) and sulfonyl (15, -CH2SO2Me, -SO2Me) > sulfinyl (14, -CH2SOMe, -SOMe). The point of attachment of the phenyl substituent was a determinant of activity for the –CH2SMe (13a–c), -CH2SOMe (14a–c) and SOMe (14d–f) isomers where the relative potency order was meta and para > ortho. Compounds in this group (13–15), unlike Bay K 8644 (EC50 = 2.3 × 10–7 M on GPILSM), did not exhibit an agonist effect on GPILSM. The meta-CH2SMe (13b), ortho-CH2SMe (13c), meta-SMe (13e), and ortho-CH2SO2Me (15c) C-4 phenyl derivatives exhibited respectable in vitro cardiac positive inotropic activities (EC50 = 1.00 × 10–6 to 7.57 × 10–6 M range) relative to the reference drug Bay K 8644 (EC50 = 7.70 × 10–7 M) in the GPLA assay. In contrast to Bay K 8644, which acts as an undesirable calcium channel agonist on smooth muscle (GPILSM), compounds 13b (IC50 = 4.11 × 10–6 M), 13c (IC50 = 2.29 × 10–5 M), 13e (IC50 = > 1.20 × 10–5 M) and 15c (IC50 = 6.22 × 10–6 M) exhibited a desirable simultaneous calcium channel antagonist effect on smooth muscle at a similar (13b, 15c), or lower (13c, 13e), concentration relative to its cardiac agonist EC50 value. Model compounds such as 13b, 13c, 13e, and 15c, that exhibit dual cardioselective agonist / smooth muscle selective antagonist activities, represent a novel type of 1,4-dihydropyridine CC modulators that offer a potential approach to drug discovery targeted toward the treatment of congestive heart failure and for use as probes to study the structure–function relationship of calcium channels. Drug Dev. Res. 51:177–186, 2000. © 2001 Wiley-Liss, Inc.

Steric Effects and Steric Inhibition of Resonance in Isopropylbenzoic Acids in the Gas Phase and in Solution

The substituent effects of the isopropyl group in 2-, 3- and 4-isopropylbenzoic acids were investigated on the enthalpies of formation, gas-phase acidities, acidities in methanol and in dimethyl sulfoxide, and on the IR spectra in tetrachloromethane. Particular attention was given to the influence of variable conformation on the observed steric effect. In contrast to 2-tert-butylbenzoic acid and similarly to 2-methylbenzoic acid, 2-isopropylbenzoic acid exists in two planar conformations in equilibrium. Due to this conformational freedom, the steric effects of the isopropyl group on the conformation of the carboxyl group or on the gas-phase acidity are relatively small, rather close to that of a methyl group. The gas-phase acidity can be qualitatively described in terms of pole/induced dipole interaction in the anion. The latter effect is observable with a lower intensity even in 3-isopropylbenzoic acid: in this action from a more remote position, the isopropyl group resembles more the tert-butyl than the methyl group. In methanol and dimethyl sulfoxide solutions, the steric effects and particularly the pole/induced dipole interaction are attenuated and effect of isopropyl acquires intermediate values between methyl and tert-butyl. Solvent effects are thus deciding for the position of the isopropyl substituent on the scale of steric substituent constants.

Fabrication of silicon field emission points for vacuum microelectronics by wet chemical etching

The fabrication of sharp silicon tips for field emission cathodes has been investigated using anisotropic and isotropic wet chemical etching techniques. Formulations of potassium hydroxide, water and alcohol are used as the anisotropic etchants. The effects of isopropyl, secondary butyl and tertiary butyl alcohols on tip formation are examined. An isotropic etchant using a mixture of hydrofluoric, nitro and acetic acids is also used to form similar cathode structures for comparison. The silicon needles formed are intended for use in vacuum microelectronic devices.

Contraction of chromosomes and depression of RNA synthesis by isopropyl N-(3-chlorophenyl) carbamate (CIPC) in Vicia faba root tip cells.

The effects of isopropyl N-(3-chlorophenyl) carbamate (CIPC) on Vicia faba root tip cells were studied. The contraction of mitotic chromosomes was conspicuously intensified by CIPC. The susceptibility to CIPC was comparatively lower in this plant than in other reported plants regarding to a growth inhibition, and the degree of contraction of chromosomes was rather time dependent but not very significantly dose dependent. Regular formation of mitotic apparatus was blocked. Autoradiography showed the depression of RNA synthesis in nucleus.

Effects of Isopropyl N-(3-Chlorophenyl) Carbamate on Suberin, Periderm, and Decay Development by Katahdin Potato Slices

1. Freshly cut Katahdin potato slices were dipped momentarily in suspensions containing 0, 5, 25, 50, 100, 500, or 1250 p.p.m. of isopropyl N-(3-chlorophenyl) carbamate (CIPC), and held in moist chambers at 60⚬ F. for 2, 4, 7, or 14 days, then rated for suberin and periderm development. 2. Suberin and periderm developed on all slices treated with 5 p.p.m. CIPC. At 25 p.p.m. periderm development was retarded, while at 50 p.p.m. and above periderm development was almost completely inhibited. Retardation of development of suberin was noted in some slices treated at the higher concentrations. 3. After being treated with various concentrations of CIPC, Katahdin potato slices were held 2, 4, 7, or 14 days at 60⚬ F., inoculated with cultures of Erwinia carotovora (L. R. Jones) Holland and then rated after 2 days for development of decay. More decay was found in slices held for shorter periods and in those treated with higher concentrations of CIPC. 4. When slices were held long enough after treatment with CIPC to allow extensive suberin development, bacterial infection was retarded or prevented, even though periderm did not develop.

Some effects of isopropyl N-(3-chlorophenyl) carbamate on respiration, water uptake and ion leakage of potato tissue

Some effects of isopropyl N-(3-chlorophenyl) carbamate on respiration, water uptake and ion leakage of potato tissue

Some Effects of Isopropyl N-(3-Chlorophenyl) Carbamate and an Alkanolamine Salt of Dinitro Ortho Secondary Butyl Phenol on Germinating Cotton Seeds

Some Effects of Isopropyl N-(3-Chlorophenyl) Carbamate and an Alkanolamine Salt of Dinitro Ortho Secondary Butyl Phenol on Germinating Cotton Seeds