Advantages and Limitations of Salmon-Gal/Tetrazolium Salt Histochemistry for the Detection of LacZ Reporter Gene Activity in Murine Epithelial Tissue

The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial β-galactosidase (Bact β-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact β-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous β-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact β-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine–potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact β-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.

Liquid-liquid extraction of ion-association complexes of cobalt(II)-4-(2-pyridylazo)resorcinol with ditetrazolium salts

The formation and liquid-liquid extraction of ion-association complexes between Co(II)-4-(2-Pyridylazo)resorcinol (PAR) anionic chelates and cations of three ditetrazolium chlorides were studied: Blue Tetrazolium chloride (BTC), Neotetrazolium chloride (NTC) and Nitro Blue Tetrazolium chloride (NBT). The optimum conditions for the formation and solvent extraction of the ion-association comlpex chelates were determined. It has been found that in the systems of Co(II)-PAR-DTS, the reactants are reacted in molar ratios 1:2:1 and the general formula of complexes was suggested. The extraction equilibria were investigated and quantitatively characterized by the equilibrium constants and the recovery factors. The analytical characteristics of the complexes were calculated.

Functional and metabolic neutrophils activity in patients with acute intestinal infections and the influence of selimakcid

Aim. To explore the functional activity of neutrophils in healthy individuals and patients with acute intestinal infections and the effect of selimakcid on it in vitro. Methods. 48 patients with acute intestinal infections aged 20 to 60 years were examined. The control group consisted of 10 healthy people of the same age. Functional and metabolic activity of neutrophils was studied by a nitro blue tetrazolium chloride test and by «BURSTTEST» (Neutrophil Oxidative Burst Test). Results. Examination of the functional and metabolic activity of neutrophils in patients with acute intestinal infections revealed increased results of spontaneous nitro blue tetrazolium chloride test in acute period of the disease. The same changes were discovered while studying the Serratia marcescens stimulated cell activity in patients with dysentery. However, in patients with salmonellosis neutrophils activity in induced nitro blue tetrazolium chloride test did not differ from that of healthy controls. The number of neutrophils synthesizing reactive oxidants recorded by «BURSTTEST» in patients corresponded to the indicators of the induced nitro blue tetrazolium chloride test and did not differ in patients with different nosologies. Studying the effect of selimakcid on neutrophils functional activity in healthy controls and patients with acute intestinal infections by nitro blue tetrazolium chloride test and «BURSTTEST» showed that pre-incubation of neutrophils with selimakcid intensified the response to Serratia marcescens. The ability of the drug to modulate the neutrophils activity in patients with acute intestinal infections was higher compared to healthy individuals. Conclusion. Increased functional activity of neutrophils associated with reduced reserve capacity was revealed in patients with salmonellosis and shigellosis of moderate severity. Selimakcid showed immunomodulatory effect, increasing the functional and metabolic potential of neutrophils in patients with acute intestinal infections in vitro.

Phenylmethoxybis(tetrazolium) ion-association complexes with an anionic indium(III) — 4-(2-pyridylazo)resorcinol chelate

Complex formation and liquid-liquid extraction were studied in systems containing indium(III), 4-(2-pyridylazo)resorcinol (PAR), phenylmethoxybis(tetrazolium) salt (MBT), water and chloroform. The following MBTs, which differ only by the number of -NO2 groups in their cationic parts, were used: 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis(2,5-diphenyl-2H-tetrazolium chloride) (Blue Tetrazolium chloride, BT), 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (Nitro Blue Tetrazolium chloride, NBT) and 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis[2,5-di(4-nitrophenyl)-2H-tetrazolium chloride] (Tetranitro Blue Tetrazolium chloride, TNBT). The composition of the formed ternary complexes was determined, In:PAR:MBT=1:2:2, and the optimum conditions for their extraction found: pH, shaking time, concentration of the reagents and the sequence of their addition. Some key constants were estimated: constants of extraction (Kex), constants of association (β) and constants of distribution (KD). BT appears to be the best MBT for extraction of the In(III)-PAR species, [In3+(OH)3(PAR)2]4−, (Log Kex=10.9, Log β=9.8, Log KD=1.12, R%=92.7%). Several additional characteristics concerning its application as extraction-spectrophotometric reagent were calculated: limit of detection (LOD = 0.12 µg cm−3), limit of quantification (LOD = 0.40 µg cm−3) and Sandell’s sensitivity (SS =1.58 ng cm−2); Beer’s law is obeyed for In(III) concentrations up to 3.2 µg mL−1 with a molar absorptivity coefficient of 7.3×104 L mol−1 cm−1 at λmax=515 nm.

Spectrophotometric Determination of Carbon Disulphide in the Workplace Air

This papre describes a simple method of carbon disulphide determination in the air of working environment in the chemical companies and plants after its absorption into aprotic N,N-dimethylformamide solvent. Carbon disulphide absorbed into aprotic solvent was transformed by using ammonium hydroxide on sulphides which were determined by spectrophotometry. 5,5′-Dithiobis(2-nitrobenzoic acid) and blue tetrazolium chloride were used as chromogenic sensing reagents. Colour-reducing products were measured at the wavelength of 500, respectively 520 nm. Detection limits for determination of carbon disulphide in the air are 0.2, respectively 0.4 mg·m−3.