Characterization of Human γδ T Lymphocytes Infiltrating Primary Malignant Melanomas

T lymphocytes are often induced naturally in melanoma patients and infiltrate tumors. Given that γδ T cells mediate antigen-specific killing of tumor cells, we studied the representation and the in vitro cytokine production and cytotoxic activity of tumor infiltrating γδ T cells from 74 patients with primary melanoma. We found that γδ T cells represent the major lymphocyte population infiltrating melanoma, and both Vδ1⁺ and Vδ2⁺ cells are involved. The majority of melanoma-infiltrating γδ cells showed effector memory and terminally-differentiated phenotypes and, accordingly, polyclonal γδ T cell lines obtained from tumor-infiltrating immune cells produced IFN-γ and TNF-α and were capable of killing melanoma cell lines in vitro. The cytotoxic capability of Vδ2 cell lines was further improved by pre-treatment of tumor target cells with zoledronate. Moreover, higher rate of γδ T cells isolation and percentages of Vδ2 cells correlate with early stage of development of melanoma and absence of metastasis. Altogether, our results suggest that a natural immune response mediated by γδ T lymphocytes may contribute to the immunosurveillance of melanoma.     

Effect of β-Propiolactone on Sendai Virus

The biological properties (infectivity, hemagglutination, hemolysis, cell fusion, neuraminidase) of Sendai virus were dissociated on the basis of sensitivity to beta-propiolactone, by freeze-thawing, by heating at different temperatures, and by adsorption-elution with formalinized chicken erythrocytes. Possible mechanisms whereby beta-propiolactone selectively destroys viral infectivity are discussed.     

Effect of protein-surfactant interactions on aggregation of β-lactoglobulin

The milk protein β-lactoglobulin (βLG) dominates the properties of whey aggregates in food products. Here we use spectroscopic and calorimetric techniques to elucidate how anionic, cationic and non-ionic surfactants interact with bovine βLG and modulate its heat-induced aggregation. Alkyl trimethyl ammonium chlorides (xTAC) strongly promote aggregation, while sodium alkyl sulfates (SxS) and alkyl maltopyranosides (xM) reduce aggregation. Sodium dodecyl sulfate (SDS) binds to non-aggregated βLG in several steps, but reduction of aggregation was associated with the first binding step, which occurs far below the critical micelle concentration. In contrast, micellar concentrations of xMs are required to reduce aggregation. The ranking order for reduction of aggregation (normalized to their tendency to self-associate) was C10-C12>C8>C14 for SxS and C8>C10>C12>C14>C16 for xM. xTAC promote aggregation in the same ranking order as xM reduce it. We conclude that SxS reduce aggregation by stabilizing the protein's ligand-bound state (the melting temperature t(m) increases by up to 10°C) and altering its charge potential. xM monomers also stabilize the protein's ligand-bound state (increasing t(m) up to 6°C) but in the absence of charged head groups this is not sufficient by itself to prevent aggregation. Although micelles of both anionic and non-ionic surfactants destabilize βLG, they also solubilize unfolded protein monomers, leaving them unavailable for protein-protein association and thus inhibiting aggregation. Cationic surfactants promote aggregation by a combination of destabilization and charge neutralization. The food compatible surfactant sodium dodecanoate also inhibited aggregation well below the cmc, suggesting that surfactants may be a practical way to modulate whey protein properties.     

Synthetic inhibitors of glucocerebroside β-glucosidase

The glucocerebrosidase of human placenta was studied with various potential inhibitors. Several compounds that resemble the lipoidal product of enzyme action, ceramide, proved to be excellent inhibitors, acting by mixed modes (primarily noncompetitively). These were N-decyl-dl-erythro-3-phenyl-2-amino-l, 3-propanediol and several p-substituted derivatives. These compounds were also highly effective in rat spleen toward glucocerebroside and p-nitrophenyl β-glucoside as substrates. The compounds were inactive toward the analogous enzyme, galactocerebrosidase of rat brain, and were slightly stimulatory toward the rat brain enzyme which makes galactocerebroside. Longer and shorter N-alkyl groups proved to be less effective. Decanoic acid amides of phenylaminopropanediol and related compounds proved to be relatively inert, although some were stimulatory. Deoxycorticosterone β-glucoside was a moderately effective noncompetitive inhibitor and is apparently hydrolyzed by a different glucosidase. p-Nitrophenyl β-glucoside was also a moderately effective inhibitor, acting by mixed modes. p-Chloromercuribenzenesulfonate was a good inhibitor, presumably acting on a sensitive cysteine residue. It is concluded that cerebrosidase contains two sensitive sites, one catalytic and the other allosteric, each containing an important anionic group and able to bind glucosides and ceramide-like compounds.     

Specificity and Selectivity of a Modified Radioligand Method for Estimating the Binding Activity of β1 Adrenergic Receptors in Human T Lymphocytes

Russian Journal of Bioorganic Chemistry - A radioligand-based method was proposed for quantifying the binding activity of β1-adrenergic receptors (ARs) on the surface of human T lymphocytes....     

Inhibitory Effect of Methyl Methanethiosulfinate on β-Glucuronidase Activity

Minced or cut-up leaves of Chinese chive (Allium tuberosum Rottler) contain thiosulfinates and their disproportionate reaction products. Among these organosulfur compounds, methyl methanethiosulfinate was found to be an uncompetitive inhibitor of β-glucuronidase. Approximately 80% of the enzyme activity was inhibited by methyl methanethiosulfinate at 50 µM, the IC₅₀ value being comparable to 3.6 µM.     

Effects of Intracellular Calcium Chelation and Pifithrin-α on Deoxynucleotide Metabolism in Human Lymphocytes

Previously, we have found that activation of deoxycytidine kinase elicited by various DNA-damaging chemical agents could be prevented by BAPTA-AM, a cell-permeable calcium chelator or by pifithrin-α, a pharmacological inhibitor of p53. Here, we show that stimulation of deoxycytidine kinase by UV-light also is calcium-dependent and pifithrin-α-sensitive in tonsillar lymphocytes, while thymidine kinase 1 activity is stabilised in the presence of BAPTA-AM. Importantly, both UV-irradiation and calcium chelation decreased the incorporation of labelled deoxycytidine and thymidine into DNA. Pifithrin-alpha dramatically reduced the labelling of both the nucleotide and DNA fractions, possibly due to inhibition of transmembrane nucleoside transport.     

Effect of the Disease-Causing R266K Mutation on the Heme and PLP Environments of Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ν(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.     

Synthetic Peptide Vaccines: Unexpected Fulfillment of Discarded Hope?

In the early eighties it was realized that the ultimate vaccine would be a synthetic peptide. Major efforts were put into the development of a synthetic vaccine for foot-and-mouth disease virus (FMDV) for which even today no alternative exists besides the classical vaccine based on inactivated virus. Despite impressive progress, a peptide vaccine that could match the classical vaccine with respect to efficacy (i.e. full protection of all animals after a single vaccination) has not materialized.This has led to the belief that synthetic vaccines were not possible. However, in the early nineties we developed a synthetic peptide vaccine for canine parvovirus that did match the classical vaccine based on inactivated virus (i.e. protected all animals). Based on the difference of FMDV (an RNA virus) and canine parvovirus (a DNA virus), we suggested that in the case of FMDV, more than one antigenic site should be used, instead of the single one used previously. In our opinion multiple sites are necessary to prevent the development of escape mutants of FMDV. Unfortunately, the additional sites of FMDV are highly discontinuous. Until recently it was impossible to reconstruct these sites in the form of synthetic peptides. In the past few years, new methods have been developed that allow recombination of such sites into synthetic molecules. If successfully applied to FMDV, synthetic peptide vaccines and many others may become feasible in the near future. Moreover, the ability to mimic complex discontinuous sites by synthetic peptides will have a major impact on the rapidly developing area of therapeutic vaccines.     

Effect of β-adrenergic stimulation of trout erythrocytes on blood viscosity

1. Both the viscosity of trout blood and its dependence on shear rate are reduced when erythrocyte β-adrenoceptors are stimulated.2. This suggests that catecholamines released during stress may decrease the resistance of nucleated erythrocytes to blood flow.     

Effect of ethionine on the synthesis of β-galactosidase inEscherichia coli

Ethionine at concentrations of 10⁻³M, 5×10⁻³M and 10⁻²M inhibits growth, both of β-galactosidase inducible ML-30 and constitutive ML-308Escherichia coli strains. The protein synthesis (measured by the incorporation of l-leucine-¹⁴C and l-aspartic-¹⁴C acid into proteins) of these strains is inhibited to the same extent as their growth. The synthesis of inducible and constitutive β-galactosidase produced by the strains ML-30 and ML-308, respectively, is considerably inhibited by ethionine.     

Effect of β-phenylethylamine on dopaminergic systems of the rat brain

The time course of changes in the dopamine concentration in dopaminergic neurons of the nigro-neostriatal and mesolimbic systems of the rat brain during 1 h after intraperitoneal injection of -phenylethylamine (100 mg/kg) was studied by quantitative fluorescence-histochemical analysis. The results showed that -phenylethylamine causes a marked fall in the dopamine level in neurons of dopaminergic systems of the brain. The dopamine level in the bodies of dopaminergic neurons changes more than in their axon terminals. The fall in the dopamine concentration in the dopaminergic systems of the brain during the first hour is irregular in character: in the terminals between 10 and 30 min and in the bodies between 30 and 45 min there is actually a temporary increase in the dopamine concentration. The rise in the dopamine concentration in the terminals coincides with a sharp fall in the dopamine level in the neuron bodies, and conversely, the fall in the dopamine concentration in the terminals after 30 min is accompanied by some increase in the dopamine concentration in the neuron bodies. The results suggest that the increase in motor activity described in the literature in animals after injection of -phenylethylamine is connected with its action on catecholaminergic, especially dopaminergic, brain systems.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 578–584, November–December, 1979.     

Effect of Crystallization State on the Gel Properties of Oleogels Based on β-sitosterol

Oleogels based on three different oils (sunflower oil, solid coconut oil and liquid coconut oil) were formulated using β-sitosterol. In general, an observed increase in crystallinity was correlated with an increase in the gel storage modulus and hardness. Addition of lecithin promoted the formation of needle-like crystals of β-sitosterol with a corresponding increase in strain tolerance and oil-trapping capacity for oleogels produced with liquid oils. However, the incorporation of β-sitosterol crystals with or without lecithin into oleogels containing solid coconut oil reduced its strain tolerance by interrupting the formation of continual radiolitic crystal structures. The use of sunflower oil (long chain fatty acids) was more favourable to the packing and growth of gelator crystals and the formation of an elastic gel, in comparison to liquid coconut oil (short chain fatty acids). Overall, the type and physical state of oil influence the formation of oil crystal network, thus affecting its gel properties. These findings allow the better understanding of β-sitosterol-based oleogels, providing opportunity to design for the application as a fat-replacer and lowering solid fat content.

     

Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity

Summary. Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl₄ in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl₄ challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl₄-induced liver injury.     

Effect of chitosan degradation on its interaction with β-lactoglobulin

Complexes between chitosan and β-lactoglobulin (β-Lg) were investigated, and their formation was found to depend on pH and ionic strength. The electrostatic attraction between the cationic polysaccharide and the negatively charged protein above its isoelectric point has been identified as the main driving force in the molecular recognition process. At low protein concentration, soluble complexes were shown to be formed, and their structural features were characterized by circular dichroism (CD) and steady-state fluorescence. Both the overall secondary structure of the protein and the local environment probed by its tryptophan residues are not affected by the presence of chitosan in the complex. Furthermore, the formation of the complex does not lead to a net stabilization of the native state of the protein over its denatured state due to formation of a similarly stable complex between the polyelectrolyte and the denatured state of the protein. The formation of coacervates between β-Lg and chitosan was also characterized as a function of average molecular weight of chitosan (subjected to ultrasonication for different periods of time: 0, 5, 15, and 30 min) by means of both turbidimetric and calorimetric techniques. The combination of turbidimetric as well as isothermal calorimetric titrations have allowed the deconvolution of two processes usually coupled in the formation of protein-polyelectrolyte coacervates: the formation of complex coacervates as the protein sites become saturated by polyelectrolyte molecules and the redissolution of the coacervates as the polyelectrolyte-to-protein ratio increases.     

Effect of preischemic β-adrenoceptor stimulation on postischemic contractile dysfunction

AimsShort periods of preischemic β-adrenoceptor stimulation protect hearts against postischemic left ventricular dysfunction. It was the aim of this study to decide whether this procedure mimics ischemic preconditioning by the generation of preischemic hemodynamic and energetic stress or whether it represents an endogenous phenomenon and to investigate the influence of age and hypertension.Main methodsIsolated rat hearts were investigated ex vivo by Langendorff perfusion and exposed to an established ischemia/reperfusion protocol (45 min no-flow ischemia and 90 min reperfusion). Left ventricular developed pressure (LVDP), rate pressure product, and ± dP/dt were analyzed.Key findingsIsoprenaline concentration dependently increased LVDP up to 40 ± 15 mm Hg (approximately EC₅₀ of 9.9 ± 0.5 nM). Isoprenaline given prior to ischemia attenuated the subsequent postischemic ventricular dysfunction (approximately EC₅₀ of 1.4 ± 0.2 pM). However, concentrations high enough to improve LVDP in normoxic hearts did not improve postischemic recovery albeit a significant reduction of hypercontraction-induced cell damage. The effect on functional recovery was attenuated by atenolol, H89, and wortmannin suggesting that β-adrenoceptor stimulation, protein kinase A, and PI 3-kinase activation are involved. The effect was conserved in hearts from 13 month old rats but lost in age-matched spontaneously hypertensive rats.SignificanceThe study identifies preischemic β-adrenoceptor stimulation as a pharmacological preconditioning protocol that does not simply mimic classical ischemic preconditioning by induction of hemodynamic or energetic stress prior to a prolonged ischemic period. The observed loss of effectiveness in hypertensives may contribute to the reduced ischemic tolerance of hypertensives.     

Effect of Phytic Acid on the Hydrolysis of Lactose with β-Galactosidase

The effects of phytate on the hydrolysis of lactose with β-galactosidases from bovine liver and Escherichia coli were investigated. The activities of both β-galactosidases were decreased to the same extent by increased concentrations of phytate. The rates of inhibition of β-galactosidase activity from E. coli in a reaction mixture containing 10 mm of phytate were 78.9% and 64.4%, respectively, in the absence of and with 4 mm of Mg^{2 +}. Therefore, it was found that the stimulatory effect of Mg²⁺ was hardly affected by the presence of phytate in the range from 2 to 10 mm. The β-galactosidase activity was also not influenced by preincubating β-galactosidase or lactose with phytate. Kinetic studies showed that the inhibition of β-galactosidase activity by phytate was of an uncompetitive type with a Ki value of 3.46 mm. Therefore, it is considered that phytate may interact with a complex of ß-galactosidase and lactose.     

Effect of Dephosphorylation on the Self-association and the Precipitation of β-Casein

The pyrolyzate of the nondialyzable melanoidin prepared from glucose-ammonia reaction system (kept in pH 5.3~6.0 during the reaction) was fractionated to volatile fraction and nonvolatile fraction. Among the volatile components, two pyridines and four alkylpyrazines were identified. On the other hand, one imidazole compound and two β-hydroxypyridines isolated from the nonvolatile fraction were identified as 4(5)-methylimidazole, 3-hydroxypyridine and 2-methyl-5-hydroxypyridine, respectively. It is inferred that these compounds are not produced by the fission of the main skeleton in the melanoidin molecule, but formed by pyrolysis of the heterocyclic compounds present as a small moiety in the melanoidin.