Quantitative estimation of newly synthesized radioactive proteins: use of minicolumns of antibody embedded in agarose gel or immobilized on Sepharose.

Immunochemical methods for the quantitative determination of radioactive proteins synthesized by chick embryo hepatocyte cultures are described. Cell culture medium containing secreted labeled serum proteins was concentrated and applied to agarose gels containing rabbit anti-chicken serum. Nonspecific binding in control gels was reduced to less than 2% of applied counts under conditions where more than 60% of the nondialyzable counts were precipitated in the presence of antibody. Labeled cytosol fructose-1,6-bisphosphatase (Fru-P₂ase) from cell sonicates was selectively adsorbed onto columns containing Sepharose-immobilized anti-chicken liver Fru-P₂ase. Radioactivity bound on these columns was eluted with 1% sodium dodecyl sulfate for electrophoretic analysis. Addition of dialyzed horse serum was used in both cases to minimize nonspecific binding. These techniques provide simple alternatives to direct immunoprecipitations in solution where nonspecific binding of radioactivity may be unacceptably high.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

Determination of non-cholesterol sterols in serum and HDL fraction by LC/MS-MS: Significance of matrix-related interferences

BackgroundNon-cholesterol sterols (NCS) are promising biomarkers for estimation of cholesterol homeostasis properties. In addition, determination of NCS in high-density lipoprotein (HDL) fraction (HDL-NCS) could provide information on cholesterol efflux. However, matrix effects interfere in liquid chromatography-mass spectrometry (LC-MS) analysis of NCS, thereby impairing the method sensitivity. The aims of this study were development, optimization and validation of LC-MS method for quantification of NCS in serum and HDL-NCS. Additionally, matrix effect interferences and methods application in individual serum samples were examined.MethodsHDL precipitating reagent was used for HDL isolation. Matrix effect was examined by comparing different surrogates by simple regression analysis. Validation was conducted according to the FDA-ICH guideline. 20 healthy volunteers were recruited for testing of method application.ResultsThe observed matrix effect was 30%, and matrix comparison showed that cholesterol was the dominant contributor to the matrix effect. Cholesterol concentration was adjusted by construction of the calibration curve for serum and HDL fraction (5 mmol/L and 2.5 mmol/L, respectively). The intraand interrun variabilities for NCSs were 4.7-10.3% for serum NCS and 3.6-13.6% for HDLNCS and 4.6-9.5% for serum NCSs and 2.5-9.8% for HDL-NCS, respectively. Recovery studies showed satisfactory results for NCSs: 89.8-113.1% for serum NCS and 85.3-95.8% for HDL-NCS.ConclusionsThe method was successfully developed and optimized. The matrix interference was solved by customising calibration curves for each method and sample type. The measurement of NCS in HDL fraction was proposed for the first time as potentially useful procedure in biomedical researches.     

Probiotic sonicates selectively induce mucosal immune cells apoptosis through ceramide generation via neutral sphingomyelinase

Background

Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect.

Methodology/Principal Findings

Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L. brevis (LB) and S. thermophilus (ST) and the non-probiotic E. coli (EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn''s disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB-induced apoptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase.

Conclusions

These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics, and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics.     

Synthesis, structure and cytotoxic activity of mixed-valent Cu(I)/Cu(II) salt containing a pyrazolone derivative as ligand

The compound [Cu(TAMEN)][Cu(NCS)₂Cl](DMF)₂ containing the Mannich base N,N′-tetra-(4-antipyrylmethyl)-1,2-diaminoethane (TAMEN), as ligand, was synthesized and characterized by conductometric, magnetic, electronic and infrared spectroscopic properties. The single-crystal X-ray structure shows the presence of two well defined complex units, [Cu(TAMEN)]²⁺ and [Cu(NCS)₂Cl]²⁻.The complex cation contains copper(II) in the elongated pseudo-octahedral environment created by the N₂O₄ donor set of TAMEN whereas, the counterion exhibits a pseudo trigonal planar geometry around the Cu(I) ion.Both, the pyrazolonic ligand and the complex have been screened for their cytotoxic activity against human tumor cell lines: glioblastoma multiforme, breast cancer, hepatoma and lung carcinoma. The nontumor cell lines MDBK (bovine kidney) and BALB/c 3T3 clone 31 (mouse embryo) were also included in the experiments. In contrast to the ligand TAMEN, the [Cu(TAMEN)][Cu(NCS)₂Cl](DMF)₂ was found to decrease in a time- and concentration-dependent manner the viability of cultured tumor (MCF-7, A549, 8MGBA, Hep G2) and non-tumor (MDBK, BALB/c 3T3) cell lines.     

Transition-metal(II) thiocyanate coordination compounds containing 4-allyl-1,2,4-triazole. Structure and magnetic properties.

The synthesis and characterisation of a series of dinuclear and polynuclear coordination compounds with 4-allyl-1,2,4-triazole are described. Dinuclear compounds were obtained for Mn(II) and Fe(II) with composition [M₂(Altrz)₅(NCS)₄], and for Co(II) and Ni(II) with composition [M₂(Altrz)₄(H₂O)(NCS)₄](H₂O)₂. The crystal structure of [Co₂(Altrz)₄(H₂O)(NCS)₄](H₂O)₂ was solved at room temperature. It crystallizes in the monoclinic space group P2₁/n. The lattice constants are a = 18.033(3) Å, b = 13.611(2) Å, c = 15.619(3) Å, β = 92.04(2)° Z = 4. One cobalt ion has an octahedrally arranged donor set of ligands consisting of three vicinal nitrogens of 1,2-bridging triazoles (CoN = 2.14–2.15 Å), one terminal triazole nitrogen (CoN = 2.12 Å) and two N-bonded NCS anions (CON = 2.08 Å). The other Co(II) ion has the same geometry, but the terminal triazole ligand is replaced by H₂O (CoO = 2.15 Å). The crystal structure is stabilised by hydrogen bonding through H₂O molecules, S-atoms of the NCS anions and the lone-pair electron of the monodentate triazole. The magnetic exchange in the Mn, Co and Ni compounds is antiferromagnetic with J-values of ?0.4 cm^?1, ?10.9 cm^?1 and ?8.7 cm^?1 respectively. The Co compound was interpreted in terms of an Ising model. For [Zn₂(Altrz)₅(NCS)₂]∞[Zn(NCS)₄], [Cu₂(Altrz)₃(NCS)₄]∞ and [Cd₂(Altrz)₃(NCS)₄]∞ chain structures are proposed. In the Cu compound thiocyanates appear to be present, bridging via the nitrogen atom, as deduced from the IR spectrum.     

Use of a simplified fluctuation test to detect and characterize mutagenesis by nitrofurans.

The capacity of whole cell sonicates of skin fibroblasts of normal individuals and patients with the autosomal recessive disease Ataxia telangiectasia (AT) to remove aerobic gamma-ray products of the 5,6-dihydroxydihydrothymine type (t₀^γ₂) from exogenous DNA substrates was investigated. All four AT strains (AT CRL 1312, AT CRL 1343, AT GM 367, AT 4BI) possessed normal capabilities to excise t₀^γ₂ from irradiated bacteriophage DNA and irradiated chromatin isolated from normal and AT-skin fibroblasts.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Trans-bis(benzo(1,3)-thiazole)bis(N,N-dimethylformamide)bis(N-thiocyanato)-cobalt(II): spectroscopy,thermal analysis and crystal structure

Disolution of Co(bzt)₂(NCS)₂ (bzt = benzo 1,3- thiazole) in dimethyl formamide (dmf) produces Co(bzt)₂(NCS)₂(dmf)₂. The stoichionmetry of the complex has been established by a combination of chemical (C, H, N) and thermal analysis. The comlex has an octahedral structure with pairs of ligands in trans configuration as well as a CoN₄O₂ coordination sphere with CoN distances of 2.185(2) Å (bzt); 2.082(2) Å (NCS) and CoN(dmf) of 2.118(2) Å. The infrared and electronic absorption spectra are consistent with this arrangement.     

Coordination chemistry of substituted [1,2,4]triazolo[1,5-a]pyrimidines with first-row transition-metal ions: Synthesis, spectroscopy and single-crystal structure analysis

A number of new coordination compounds with transition-metal salts and triazole-based heterocyclic ligands is described. The ligands used are disubstituted [1,2,4]triazolo[1,5-a]pyrimidines, with as substituents: ethyl, methyl and phenyl, and in addition a 2-methylthio-dimethyl ligand was used (sdmtp). To determine the structures of the coordination compounds in a number of cases 3D crystal structure determinations have been carried out, i.e. for [Fe(NCS)₂(detp)₃(H₂O)], [Co(NCS)₂(sdmtp)₂(H₂O)], [ZnBr₂(fmtp)₂], [Ni(NCS)₂(detp)₃(CH₃OH)] and [Fe(NCS)₂(fmtp)₂(H₂O)₂](fmtp). The latter compound is quite unusual as it contains an uncoordinated fmtp ligand in the crystal lattice with special packing features. The ligands and the coordination compounds have been further characterized by NMR, IR and LF spectra, as well as by C, H, N element analyses. The coordination around the metal varies from 4 (Zn), via 5 (Co) to 6 (for Ni and Fe).     

Production of IgG and IgM from murine hybridomas adapted to newborn calf serum

Conclusion Application of NCS as the serum supplement in media has been shown to be effective for antibody production for the cell lines tested. High levels of antibody titers in the supernatant can be obtained in a fedbatch culture. The secretion rates in the fedbatch cultures are within an order of magnitude of the optimalin vivo secretion rate of 1 g/10⁶cell·hr. Replacement of FCS by NCS, where possible, should be considered as an economically viable option for serum supplemented large scale cell culture.     

Effects of 90 min of manual repetitive work on skin temperature and median and ulnar nerve conduction parameters: A pilot study in normal subjects

To test whether the influence of manual activity should be considered when interpreting the results of nerve conduction study (NCS) of the upper limbs performed during work shifts, we evaluated the short-term effect of 90-min repetitive manual work on NCS parameters. Twenty-eight healthy volunteers underwent NCS of the dominant limb at the end of an interview (T₀), after a 30-min rest in sitting position (T₁) and after performing a standardized 90-min manual task (T₂). The task was designed to simulate typical assembly and packing activities. No significant differences were observed for skin temperature (Ts) and NCS parameters between T₀ and T₁. Significantly (p < 0.001) higher Ts mean values were found at T₂ as compared to the previous tests for both females and males. The regression analysis showed an association between temperature variation and nerve conduction velocity values for the median and ulnar nerve at T₂ as compared to T₁. In females, a reduction of the mean sensory nerve action potential (SNAP) amplitude at T₂ was recorded, whereas an opposite trend was observed among males. Manual work is able to influence hand Ts and to modify NCS parameters. SNAP amplitudes changes suggest gender differences in peripheral nerve characteristics that deserve further investigation.     

On the existence of tris 2,2′-biquinoline complexes of first transition series metal ions in the solid state. Spectra and magnetic characterization of [Fe(II)(2,2′-biquinoline)3] (NCS)2 and some related pseudo-tetrahedral ferrous compounds

Double benzo substitution cis to the nitrogen atoms of 2,2′-bipyridine results in 2,2′-biquinoline, hereafter, biq. Numerous examples of tris 2,2′-bipyriidne metal species are known, but to out knowledge, there have been no previous reports of tris biq metal species. Herein, we report the first isolation of what appears to be a tris biq species, Fe(biq)₃(NCS)₂, in the solid state obtained from a water-acetone solution as an analytically pure, pale blue-gray solid. Zero and high field Mössbauer, optical (near infrarcd-visible), and infrared spectra as weil as cryomagnetic data clearly show that Fe(biq)₃(NCS)₂ contains a pseudo-octahedral high spin iron(II) chromophore. Several possibilities are proposed for the structure of Fe(biq)₃(NCS)₂ including some with unidentate biq. The weak field, highly hindered environment of the complex is such that it readily undergoes facile loss of two moles of ligand simply on mixing the solid complex with a variety of organic solvents. The resulting neutral buff-colored iron(II) complex, Fe(biq)(NCS)₂, is a pseudo-tetrahedral monomer which can also be prepared by the direct reaction of an ethanolic solution of Fe(NCS)₂ with a benzene solution of biq in a 1:1 stoichiometry. The spectral and cryomagnetic properties of Fe(biq)- (NCS)₂ as well as those for the related pseudo-tetrahedral compounds Fe(biq)Cl₂, Fe(2,9-dimethyl-l,10-phenanthroline)X₂ (X=CI^-, Br^-, I^-, NCS^-) and Fe(2,9-dimethyl-4,7-di-phenyl- 1,10-phenanthroline)Cl₂, are reported as weil. In particular, the latter pseudo-tetrahedral systems are found to exhibit a novel, complex range of slow paramagnetic behavior depending on T, H_o and specific coordinated anion. This behavior correlatcs with low temperature susceptibility data.     

Spectroscopic,viscositic and electrochemical studies of DNA interaction with a novel mixed-ligand complex of nickel (II) that incorporates 1-methylimidazole and thiocyanate groups

A novel mixed-ligand nickel(II) complex that contains 1-methylimidazole and thiocyanate, Ni(NCS)₂(Mim)₄ (Mim = 1-methylimidazole), was synthesized and its structure was determined by X-ray crystallography, IR spectrum and elemental analysis, etc. Its DNA-binding properties were studied by electronic absorption spectral, viscositive and electrochemical measurements. The absorption spectral and viscositive results suggest that the nickel(II) complex binds to DNA via partial intercalation. The addition of DNA results in the decrease of the peak current of the nickel(II) complex proved their interaction. The slight differences of peak profiles and electrochemical parameters between free and DNA-bound Ni(NCS)₂(Mim)₄ showed the formation of an electrochemical inactive complex between Ni(NCS)₂(Mim)₄ and DNA. The binding site and binding constant of the complex to DNA were determined by electrochemical titration method.     

Synthesis, characterization and properties of iron(II) complexes with a series of tripodal ligands based on the parent ligand tris(2-pyridylmethyl)amine

Iron(II) complexes of the type [Fe(L)(NCS)₂] with the tripodal ligand apme (apme = N¹-(2-aminoethyl)-N¹-(2-pyridyl-methyl)-1,2-ethanediamine) as well as with its derivatives were prepared and structurally characterized. The bond distances thus obtained showed that all complexes investigated were high-spin at the respective temperature. Furthermore [Fe(Me₄apme)(NCS)₂] was analyzed using Mößbauer spectroscopy that showed that this complex remains in its high-spin state over the entire temperature range.     

Preparation and crystal structure of manganese(II) isothiocyanate tetrahydrate

Crystals of Mn(NCS)₂·4H₂O were isolated from an aqueous solution obtained by mixing solutions of barium thiocyanate and manganese(II) sulphate tetrahydrate. The crystals are monoclinic with a = 7.827(7), b = 9.208(1), c = 7.456(5) Å, β = 112.57(5)°, space group P2₁/c. The structure consists of discrete centrosymmetric trans-[Mn(NCS)₂(H₂O)₄] octahedra linked by hydrogen bonds.     

Glycosidase and cumulus dispersal activities of acrosomal extracts from opossum (marsupial) and rabbit (eutherian) spermatozoa

Opossum and rabbit sperm sonicates dispersed the mouse cumulus oophorus in vitro at the same rate on a per sperm basis, despite much higher activities of the glycosidic acrosomal enzymes N-acetylhexosaminidase (350 ×) and arylsulphatase (40 ×) in the opossum preparation. Activities of another glycosidase, hyaluronidase, and the protease acrosin where similar in sperm extracts from both species. However, specific inhibitors of N-acetylhexosaminidase (iodoacetate) and arylsulphatase (SO₄^2?, PO₄^3?) markedly reduced the rate of cumulus dispersal by rabbit sperm sonicates. These findings suggest that, while hyaluronidase action probably represents the rate-limiting (slow) step, several glycosidases act together to disperse the cumulus and in the passage of the fertilizing spermatozoon through it in eutherian mammals. The likely role of these acrosomal enzymes in the marsupial, where the ovulated oocytes lack a cumulus oophorus, is more uncertain.     

Synthesis, crystal structures and properties of iron(III) complexes exhibited both high-spin and low-spin states within the crystal lattice

The reaction of FeCl₃ · 6H₂O, potassium hydrotris(pyrazolyl)borate (KTp) and KSCN gives [FeTp₂][TpFe(NCS)₃] (1) and [FeTp₂]₃[Fe(NCS)₆] (2), respectively. The bond lengths and angles indicate that both complexes have [FeTp₂]⁺ cations where Fe(III) ions are in typical low-spin state, and in counter ions, [TpFe(NCS)₃]⁻ for 1 and [Fe(NCS)₆]³⁻ for 2 are both in high-spin state. Variable temperature magnetic susceptibility and ESR results also show that there are double spin states of iron(III) ions within the crystal lattice of both compounds.     

Linkage isomerism in the metal complex hexa(thiocyanato)rhenate(IV): Synthesis and crystal structure of (NBu4)2[Re(NCS)6] and [Zn(NO3)(Me2phen)2]2[Re(NCS)5(SCN)]

The linkage isomers [Re(NCS)₆]^2? and [Re(NCS)₅(SCN)]^2? are obtained by the reaction of [ReBr₆]^2? with NCS^? in dimethylformamide. Some differences in the chemical behavior allowed their separation and structural characterization in the form of (NBu₄)₂[Re(NCS)₆] (1) and [Zn(NO₃)(Me₂phen)₂]₂[Re(NCS)₅(SCN)] (2), respectively (Bu = n-C₄H₉ and Me₂phen = 2,9-dimethyl-1,10-phenanthroline).     

Reptiles as a Source of Salmonella O48��Clinically Important Bacteria for Children: The Relationship Between Resistance to Normal Cord Serum and Outer Membrane Protein Patterns

Bacteria of the Salmonella O48 somatic antigen group are clinically important strains causing intestinal dysfunction and diarrhoea, especially in children. The susceptibility of Salmonella O48 strains containing sialic acid (N-acetylneuraminic acid (NeuAc)) in lipopolysaccharide (LPS) to the bactericidal action of normal cord serum (NCS) was determined. The authors' previous results published in Microbial Ecology in 2010 indicated that neither the presence of NeuAc in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of normal human serum (NHS), and that the presence of NeuAc in the LPS structure is not sufficient to block the activation of the alternative pathway of complement in NHS. The current results showed that the tested strains showed various sensitivities also to the bactericidal action of NCS. The authors postulate that the presence of certain outer membrane proteins (OMPs) are characteristic of the resistant and sensitive phenotypes of Salmonella O48 strains. To establish a possible relationship between resistance to NCS and OMPs band patterns, ten Salmonella O48 strains were studied as follows: susceptibility to the bactericidal effect of NCS, the mechanisms of NCS activation and OMP band patterns obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.