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At least 433 records · Page 24Linked to original sources
PubMed1994

Expression, purification, and kinetic characterization of the mannitol transport domain of the phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli. Kinetic evidence that the E. coli mannitol transport protein is a functional dimer.

The overexpression of the membrane-bound C domain of the mannitol transport protein EIIMtl of Escherichia coli has been achieved. This protein, IICMtl, consisting of the first 346 amino acids, was purified from membrane vesicles and still bound mannitol with a high affinity. Gel filtration experiments showed that purified IICMtl was a dimer, confirming that the interaction within the EIIMtl dimer occurs between the membrane-bound portions of the protein. IICMtl in combination with a chimeric protein consisting of the membrane-bound EIIGlc C domain and the cytoplasmic EIIMtl BA domain could restore both phosphoenolpyruvate-dependent phosphorylation and mannitol/mannitol-P exchange activity. The interaction in this complex was comparable to that of IICMtl with soluble IIBAMtl in as much as there appeared to be no specific interaction between IICMtl and the membrane-bound EIIGlc C domain; the Km of IICMtl for the chimer was so low that saturation could not be achieved. In contrast, a very high affinity with a Km of 2 nM was measured between purified IICMtl and purified EIIMtl. This interaction was manifested in a IICMtl-dependent stimulation of the EIIMtl catalyzed phosphoenolpyruvate-dependent mannitol phosphorylation reaction and the mannitol/mannitol-P exchange reaction. The high affinity of IICMtl for the wild type enzyme can be explained by the formation of heterodimers consisting of a IICMtl monomer and an EIIMtl monomer which interact at the level of the membrane-bound domains. The 2-fold increase in mannitol phosphorylation activity of the hetero- versus homodimer is an indication that the individual subunits in the homodimer are functionally coupled and work at only half their maximum rate. It is known that the EIIMtl dimer, but not the monomer, catalyzes the mannitol/mannitol-P exchange reaction. Since the heterodimer also catalyzes this reaction, it appears that only one functional B domain is required per dimer.

Base Sequence↗
PubMed1994

[Kinetics of luciferase gene expression from fireflies Photinus pyralis and Luciola mingrelica in Escherichia coli cells. I. Kinetics of the transformation of recombinant luciferase into enzymatically-active conformers].

The Photinus pyralis and Luciola mingrelica luciferases genes expression has been studied on the pME61 or pJG lambda plasmids with a thermoinducible lambda Pr promoter in the E. coli strain CA using three independent methods: SDS gel electrophoresis to quantify the synthesized luciferase protein, EIA to quantify the enzyme native conformer and activity measurements. The cultures were incubated by the temperature schemes 28 degrees-42 degrees-21 degrees C and 28 degrees-21 degrees C. The maximal amount of the Ph. pyralis protein reached 4.5%, while that of L. mingrelica luciferase was 4.1% of the total amount of the cellular proteins after 10-hr incubation. The amount of the native conformer and the luciferase activity began to grow after a long induction period, reaching the maximal level by hour 50-60 after the thermoinduction. The increase in the enzyme activity correlated well with the increase in the ATP content in the cell. The observed "low-temperature induction" of the enzyme activity is interpreted as a protein transition to an active conformation. This transformation is considerably behind the protein biosynthesis process. Intracellular metabolic reactions have been shown to play an active part in these conformational changes.

Animals↗
PubMed2003

Cell kinetics-dependent antitumor effect of irinotecan hydrochloride induced by the synchronizing effect of hydroxyurea: cell kinetics and dosing time.

Influence of hydroxyurea (HU) on the antitumor effect of irinotecan hydrochloride (CPT-11) was investigated in ICR male mice transplanted with sarcoma 180 cells (S-180). A single dose of CPT-11 (100 mg/kg) was injected at various times after a single dose of HU (300 mg/kg). The relative tumor weight varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The higher antitumor effect of CPT-11 was observed when DNA synthesis of S-180 cells increased (20 hr), and the lower effect was observed when the DNA synthesis decreased (0 hr). The loss of body weight also varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The toxicity of CPT-11 was higher when the inhibitory effect of HU on DNA synthesis of bone marrow cells was stronger (15 hr), and the lower toxicity was observed when the inhibitory effect was not observed (0 hr). The plasma SN-38 concentration at 2 hr after CPT-11 injection was higher at 20 hr after HU injection than at 0 hr after HU injection. The difference in plasma esterase activity between 0 hr and 20 hr after HU injection was regarded as the mechanism underlying the dosing time-dependent difference of the SN-38 concentration. These experiments suggest that HU can produce a different phase of cell cycle between tumor cells and normal cells. This leads to increase the antitumor effect of CPT-11 without increasing the adverse effect of the drug. It is essential to consider the dosing time in the two-drug combination therapy.

Animals↗
PubMed2004

Gas-phase kinetics and mechanism of the reactions of protonated hydrazine with carbonyl compounds. Gas-phase hydrazone formation: kinetics and mechanism.

The gas-phase reactions of protonated hydrazine (hydrazinium) with organic compounds were studied in a selected ion flow tube-chemical ionization mass spectrometer (SIFT-CIMS) at 0.5 Torr pressure and approximately 300 K and with hybrid density functional calculations. Carbonyl and other polar organic compounds react to form adducts, e.g., N(2)H(5)(+)(CH(3)CH(2)CHO). In the presence of neutral hydrazine, aldehyde adducts react further to form protonated hydrazones, e.g., CH(3)CH(2)CH[double bond]HNNH(2)(+) from propanal. Using deuterated hydrazine (N(2)D(4)) and butanal, we demonstrate that the gas-phase ion chemistry of hydrazinium and carbonyls operates by the same mechanisms postulated for the reactions in solution. Calculations provide insight into specific steps and transition states in the reaction mechanism and aid in understanding the likely reaction process upon chemical or translational activation. For most carbonyls, rate coefficients for adduct formation approach the predicted maximum collisional rate coefficients, k approximately 10(-9) cm(3) molecule(-1) s(-1). Formaldehyde is an exception (k approximately 2 x 10(-11) cm(3) molecule(-1) s(-1)) due to the shorter lifetime of its collision complex. Following adduct formation, the process of hydrazone formation may be rate limiting at thermal energies. The combination of fast reaction rates and unique chemistry shows that protonated hydrazine can serve as a useful chemical-ionization reagent for quantifying atmospheric carbonyl compounds via CIMS. Mechanistic studies provide information that will aid in optimizing reaction conditions for this application.

Journal Article↗
PubMed1981

Flow cytometry as a tool for the study of cell kinetics in skin 2. Cell kinetic data in psoriasis.

Flow cytometry was used to measure the DNA content of epidermal keratinocytes from psoriatic patients. Gross deviations were found in the lesions and minor but significant changes in the uninvolved skin. Statistical analysis revealed that the rather large variation in the DNA distribution of the lesions was due to inter-individual differences rather than to intra-individual differences. The duration of the S-phase seemed to be prolonged in the lesion, but the length of the overall cell cycle might be of the same order as that of normal skin.

Analysis of Variance↗