Interrelation between HeLa-S3 cell transfection and hemolysis in red blood cell suspension using pulsed ultrasound of various duty cycles

We have studied the in vitro transfection of a plasmid DNA with the lacZ gene to HeLa-S3 cells and hemolysis in a red blood cell (RBC) suspension under pulsed ultrasound with duty cycles of 10, 20 and 30% using a digital sonifier at a frequency of 20 kHz and an intensity of 6.2 W/cm² on the surface of a horn tip. Cultured HeLa-S3 cells in suspension were exposed to pulsed ultrasound for an apparent exposure time t from 0 to 60 s. HeLa-S3 viability decreased as a single exponential function of the total exposure time t=t with a common time constant =3.8 s for three duty cycles. Transfection was evaluated by counting the number of -galactosidase(-Gal)-positive cells relative to the total number of cells. Pulsed ultrasound provided an enhanced transfer of the -Gal plasmid to HeLa-S3 cells, 3.4-fold as compared with that in the case of the control. The optimal transfection efficiencies were 0.75, 0.80 and 0.74% near t= with =10, 20 and 30%, respectively. The number ratio of -Gal-positive cells to the surviving cells after exposure increased with t according to a modified logistic equation. The degree of hemolysis also increased exponentially with t at a time constant =₀/ for the RBC suspension in physiological saline at a hematocrit concentration of 0.5% with ₀=0.9 s. Thus the total exposure time for the optimal transfection efficiency was , that is, nearly four times of ₀. Hemolysis in the RBC suspension may be a useful model for determining optimal transfection by pulsed ultrasound of various duty cycles.     

An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.     

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.     

Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability

The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.     

A bivalent single-chain Fv fragment against CD47 induces apoptosis for leukemic cells

We constructed a single-chain antibody fragment (scFv) of murine monoclonal antibody, MABL, which specifically bound to human CD47 (hCD47) and induced apoptosis of the leukemic cells. The scFv of MABL antibody with a 15-residue linker (MABL scFv-15) formed both dimer (Mr 50 kDa) and monomer (Mr 25 kDa). Both MABL scFv-15 dimer and monomer had binding activity for hCD47. MABL scFv-15 dimer strongly induced apoptosis of hCD47-introduced mouse leukemic cells in vitro and exhibited anti-tumor effect in a myeloma transplanted mice model. However, MABL scFv-15 monomer scarcely exhibited these activities. These results strongly demonstrate that the ligation of CD47 antigen by two antigen-binding sites of MABL dimer is needed for inducing apoptosis. The parent MABL antibody caused hemagglutination due to the CD47 expressed on erythrocytes. Interestingly, MABL scFv-15 dimer did not cause hemagglutination. This apoptosis-inducing dimer appears to be a lead candidate for novel leukemic therapy.     

Signals from intra-abdominal fat modulate insulin and leptin sensitivity through different mechanisms: neuronal involvement in food-intake regulation

Intra-abdominal fat accumulation is involved in development of the metabolic syndrome, which is associated with insulin and leptin resistance. We show here that ectopic expression of very low levels of uncoupling protein 1 (UCP1) in epididymal fat (Epi) reverses both insulin and leptin resistance. UCP1 expression in Epi improved glucose tolerance and decreased food intake in both diet-induced and genetically obese mouse models. In contrast, UCP1 expression in Epi of leptin-receptor mutant mice did not alter food intake, though it significantly decreased blood glucose and insulin levels. Thus, hypophagia induction requires a leptin signal, while the improved insulin sensitivity appears to be leptin independent. In wild-type mice, local-nerve dissection in the epididymis or pharmacological afferent blockade blunted the decrease in food intake, suggesting that afferent-nerve signals from intra-abdominal fat tissue regulate food intake by modulating hypothalamic leptin sensitivity. These novel signals are potential therapeutic targets for the metabolic syndrome.     

Determination of thiopurine S-methyltransferase (TPMT) activity by comparing various normalization factors: reference values for Estonian population using HPLC-UV assay

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6 ng/ml/h. For heterozygous individuals (n = 18), TPMT activity was 48.1 +/- 11.7 ng/ml/h. Wild type individuals (n = 81) revealed significantly higher TPMT activity 79.3 +/- 20.7 ng/ml/h (P < 0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required.