The antiviral drug ribavirin is a selective inhibitor of S-adenosyl-L-homocysteine hydrolase from Trypanosoma cruzi

Ribavirin (1,2,4-triazole-3-carboxamide riboside) is a well-known antiviral drug. Ribavirin has also been reported to inhibit human S-adenosyl-L-homocysteine hydrolase (Hs-SAHH), which catalyzes the conversion of S-adenosyl-L-homocysteine to adenosine and homocysteine. We now report that ribavirin, which is structurally similar to adenosine, produces time-dependent inactivation of Hs-SAHH and Trypanosoma cruzi SAHH (Tc-SAHH). Ribavirin binds to the adenosine-binding site of the two SAHHs and reduces the NAD(+) cofactor to NADH. The reversible binding step of ribavirin to Hs-SAHH and Tc-SAHH has similar K(I) values (266 and 194 microM), but the slow inactivation step is 5-fold faster with Tc-SAHH. Ribavirin may provide a structural lead for design of more selective inhibitors of Tc-SAHH as potential anti-parasitic drugs.     

A sensitive and specific HPLC method for the determination of total pentosidine concentration in plasma

Pentosidine is an advanced glycation end-product (AGE) appearing when arginine and lysine residues in proteins are cross-linked with carbonyl derivatives. This paper presents an improved method for the synthesis of pentosidine and reversed-phase chromatography of this substance with fluorometric detection that enables sensitive (0.01 pmol/mg protein) and specific determination of pentosidine in plasma. Separation is done twice on the same C(18) Vydac 218TP54 column, first with trifluoroacetic acid and next with heptafluorobutyric acid as ion pair. The inter-day coefficient of variation is 6.4% at pentosidine concentration in plasma of 25 pmol/mg protein and 8% at 1.7 pmol/mg protein. Spectral properties of pentosidine exploited during identification of the substance with UV absorption and fluorescence detectors are described. Maximum of absorbance was observed at 325 nm, maximum fluorescence at lambda(ex)/lambda(em)=330/373 nm. The method may prove useful for the study of processes associated with generation and accumulation of pentosidine in the body as a marker of AGE production in healthy subjects and patients with chronic renal failure.     

Evaluation of the Influence of Magnetic Field on Female Users of an Induction Hob in Ergonomically Sound Exposure Situations

The hypothesis being tested was that the exposure of female workers to the electromagnetic field (EMF) emitted by an induction hob (IHb) meeting public exposure limitations (evaluated according to EN/IEC 62233) is also compliant with European Directive 2013/35/EU on workers’ protection. The electric field induced in three female models in a realistic ergonomically comfortable posture near IHb was evaluated using numerical models of 25 kHz EMF sources (IHb covered by cooking vessels). It was found that, in analyzed ergonomically comfortable exposure situations, the electric field induced in the user's body may exceed public and workers’ limits when the vessels do not match the dimensions of IHb's heating zone. This can even be the case when IHb complies with Conformité Européenne labeling requirements (i.e. EMF exposure falls below public limits 30 cm away from IHb edge). In the 36 exposure scenarios analyzed, statistically significant differences were found when the distances from IHb and vessel dimension, and the height and body mass index of models in exposure scenarios varied, but not between the use of models of pregnant and nonpregnant women. The use of IHb complying with European requirements on general public protection does not ensure that EMF exposure to workers complies with the relevant limits. Adequate protection measures need to address these occupational environmental hazards. © 2020 Bioelectromagnetics Society.     

Determination of the pattern of acetylation of chitosan samples: Comparison of evaluation methods and some validation parameters

Chitosan is a linear copolymer consisting of glucosamine and N-acetylglucosamine units. Its specific biological features make it a very attractive biomaterial for diverse applications in both pharmaceutics and medicine. The pattern of acetylation of chitosan samples (P_A) can strongly influence their biomedical activities. So far, three methods of determining the P_A value of chitosan samples, based on NMR spectroscopy, have been developed. Chitosans with a degree of acetylation (F_A) from 0.02 to 0.48 were used to compare these methods. The most suitable and specific method of P_A determination was ¹³C NMR based on the intensities of carbon C-5 signals. Some validation parameters—specificity, sensitivity, precision (repeatability and reproducibility)—were established. The intra- and inter-day precision of this method depended on the degree of acetylation of the chitosan samples. It was found to be specific, sensitive, repeatable and reproducible.     

Extracellular matrix components in uterine leiomyoma and their alteration during the tumour growth

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.     

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.     

Superiority of Trans-Oral over Trans-Nasal Sampling in Detecting Streptococcus pneumoniae Colonization in Adults

The human nasopharynx is the main reservoir for Streptococcus pneumoniae. We applied conventional and molecular methods to determine the prevalence of S. pneumoniae nasopharyngeal colonization in adults. Paired trans-orally and trans-nasally obtained nasopharyngeal samples from 268 parents of 24-month-old children were assessed for pneumococcal presence. Parents were classified as colonized when live pneumococci were recovered from either sample cultured on medium selective for S. pneumoniae. Of the 52 (19%) colonized parents 49 (18%) were culture-positive in trans-nasal and 10 (4%) in trans-oral samples. Bacterial growth was harvested from these cultures, DNA isolated and tested by quantitative-PCR (qPCR) targeting lytA and piaA genes specific for S. pneumoniae. A sample was considered positive if signals for both genes were detected. Altogether 105 (39%) individuals were classified as positive for pneumococcus by qPCR including 50 (19%) in trans-nasal and 94 (35%) in trans-oral settings. Although significantly more trans-nasal compared to trans-oral samples were culture-positive for S. pneumoniae at the primary diagnostic step (p<0.001) the opposite was observed in qPCR results (p<0.001). To confirm the presence of live pneumococcus in samples positive by qPCR but negative at the initial diagnostic step, we serially-diluted cell harvests, re-cultured and carefully examined for S. pneumoniae presence. Live pneumococci were recovered from an additional 43 parents including 42 positive in trans-oral and 4 in trans-nasal samples increasing the number of individuals culture- and qPCR-positive to 93 (35%) and positive by either of two methods to 107 (40%). There were significantly more trans-oral than trans-nasal samples positive for pneumococcus by both culture and qPCR (n = 71; 27%; vs. n = 50; 19%; p<0.05). Our data suggest that pneumococcal colonization is more common in adults than previously estimated and point towards the superiority of a trans-oral over a trans-nasal approach when testing adults for colonization with S. pneumoniae.     

Further development of local IL-2 therapy of cancer: multiple versus single IL-2 treatment of transplanted murine colon carcinoma

We have compared the effect of one and up to four local IL-2 treatments of transplanted MC38 colon carcinoma. A single IL-2 treatment prolonged the survival time (p=0.015), but no cure was obtained. One local IL-2 treatment inhibited tumor growth for about 1 week. After the start of tumor regrowth, a further IL-2 injection was given. After four IL-2 injections 6 out of 13 mice were cured. Histological studies show that IL-2 induced a local vascular leakage syndrome leading to massive peritumoral edema and subsequent necrosis of tumor tissue. IL-2 also attracted infiltrating cells, mainly macrophages. Subsequent IL-2 injections led to complete tumor regression. We believe that the combination of necrotic tumor debris and the IL-2–induced macrophage reaction enhanced a tumor-specific immune response. This local IL-2 application was not toxic.     

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells

Identical time-resolved fluorescence measurements with ~ 3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI–LHCI complexes and PSI–LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6–8 ps, 25–30 ps, 166–314 ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~ 90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI–LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12–15 ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI–LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.