Sulfhydryl Groups Modulate the Allosteric Interaction Between Glycine Binding Sites at the Inhibitory Glycine Receptor

We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from approximately 0.8 to values significantly above 1 (approximately 1.2-1.4) in membranes pretreated with the disulfide-reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37 degrees C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl-alkylating agent N-ethylmaleimide or fluorescein-maleimide decreased nH values to approximately 0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or beta-alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and gamma-aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Marine plant harvest in Portugal

This work brings together the scattered information on marine plant harvests and the colloid extraction industry in Portugal, as an initial contribution to the improvement of resource management. The first phase of exploitation of marine plant resources started prior to the 14th century, with the gathering and sale of storm-tossed seaweeds for fertilizer. The harvest of seagrasses and algae at Ria de Aveiro was of great economic importance. The second phase of resource exploitation began with the wider scale harvest of agarophyte species for colloid extraction. Portugal is at present the third largest harvester of the agarophytes Gelidium and Pterocladia (2500 t annually), and it is the fifth largest agar producer (350 t annually). Other colloid-producing species, including Chondrus crispus and Mastocarpus stellatus, are also harvested for export. The total agarophyte landings, agar production and income from agar exports is far below the maximum levels attained in the early 1970s. The status of stocks in each different harvest zone on the continental coast and the Azores is examined. Although there is an effective management structure for the Portuguese marine plant resource, research is needed to provide a sound biological basis for management. author for correspondence     

Immunohistochemical expression of p16INK4a and bcl-2 according to HPV type and to the progression of cervical squamous intraepithelial lesions.

Inactivation of the cell cycle inhibitor gene p16MTS1 seems to be involved in human papillomavirus (HPV)-related carcinogenesis because E6 and E7 oncoproteins may impair p16INK4a and, indirectly, bcl-2 functions. In this study, we analyzed the role of immunohistochemical expression of p16INK4a and bcl-2 in HPV-infected cervical biopsies as prognostic markers of the progression of squamous intraepithelial lesion (SIL). Sixty-five cervical biopsies were stratified into two subgroups according to the second biopsy: 27 of them maintained a low-grade (LG)-SIL diagnosis, and 38 progressed from LG-SIL to high-grade (HG)-SIL. p16INK4a and bcl-2 quantitative expression levels were measured by the immunoperoxidase method. PCR-DNA techniques were used to detect and type HPV. The Wilcoxon and Fisher exact tests were employed for the statistical analysis. In the group with an LG-SIL diagnosis at the second biopsy, no significant associations were found between p16INK4a and bcl-2 expression and presence of HPV16/18. In the group that progressed to HG-SIL, a significant association was observed between p16INK4a overexpression and HPV16/18 presence (p=0.021), but none with bcl-2 levels. It is concluded that immunohistochemical bcl-2 expression may not be useful for predicting the progression of HPV-related SIL. In contrast, p16INK4a overexpression seemed to be associated with HPV 16 and 18, suggesting that it may be a good marker for predicting SIL progression.     

Improvements in obtaining New World Leishmania sp from mucosal lesions: Notes on isolating and stocking parasites

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.     

π-π stacking tackled with density functional theory

Through comparison with ab initio reference data, we have evaluated the performance of various density functionals for describing pi-pi interactions as a function of the geometry between two stacked benzenes or benzene analogs, between two stacked DNA bases, and between two stacked Watson-Crick pairs. Our main purpose is to find a robust and computationally efficient density functional to be used specifically and only for describing pi-pi stacking interactions in DNA and other biological molecules in the framework of our recently developed QM/QM approach "QUILD". In line with previous studies, most standard density functionals recover, at best, only part of the favorable stacking interactions. An exception is the new KT1 functional, which correctly yields bound pi-stacked structures. Surprisingly, a similarly good performance is achieved with the computationally very robust and efficient local density approximation (LDA). Furthermore, we show that classical electrostatic interactions determine the shape and depth of the pi-pi stacking potential energy surface.     

Enolase from Streptococcus sobrinus is an immunosuppressive protein

A strategy of Streptococcus sobrinus, a major agent of dental caries, to survive and colonize the host consists of the production of a protein that suppresses the specific antibody responses. We have cloned the gene coding for a protein with immunosuppressive activity. It contains an open reading frame of 1302 base pairs encoding a polypeptide with 434 amino acid residues and a molecular mass of 46910 Da. The gene product is homologous to enolases from several organisms. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in a fluoride-sensitive enzymatically active form. Pretreatment of mice with the S. sobrinus recombinant enolase suppresses a primary immune response against T-cell dependent antigens. This immunosuppressive effect is specific to the antigen used in the immunization, as it is not observed when the immune response against other antigens is analysed. Furthermore, the S. sobrinus recombinant enolase stimulates an early production of interleukin-10, an anti-inflammatory cytokine, and not the pro-inflammatory cytokine IFN-gamma. These observations indicate that enolase acts in the suppression of the specific host immune response against S. sobrinus infection.     

Spectroscopic and electrochemical characterization of gold(I) and gold(III) complexes with glyoxaldehyde bis(thiosemicarbazones): cytotoxicity against human tumor cell lines and inhibition of thioredoxin reductase activity

Complexes [Au(2)(H(2)Gy3DH)(2)]Cl(2) (1), [Au(H(2)Gy3Me)]Cl(3) (2) and [Au(H(2)Gy3Et)]Cl(3) (3) were obtained with glyoxaldehyde bis(thiosemicarbazone) (H(2)Gy3DH) and its N(3)-methyl (H(2)Gy3Me) and N(3)-ethyl (H(2)Gy3Et) derivatives. The bis(thiosemicarbazones) and their gold(I) and gold(III) complexes exhibited anti-proliferative activity against HL-60, Jurkat (leukemia) and MCF-7 (breast cancer) cells at 10 μmol L(-1). Complex (2) was able to in vitro inhibit thioredoxin reductase (TrxR) activity, which suggests that inhibition of TrxR could be part of its mechanism of action.     

Regulation of the Na+-Dependent Glutamate/Aspartate Transporter in Rodent Cerebellar Astrocytes

The regulation of the Na⁺-dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with l-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca²⁺/ diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [³H]-d-aspartate uptake. This reduction was dose- and time-dependent and sensitive to PKC inhibitors. Furthermore, the l-glutamate–dependent [³H]-d-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the l-glutamate effect. In sharp contrast, in cortical astrocytes, both l-glutamate and TPA pre-exposure result in an augmentation of the [³H]-d-aspartate uptake. These findings suggest that the Na⁺-dependent glutamate uptake GLAST undergoes a region-specific regulation.     

AnthropoAge,a novel approach to integrate body composition into the estimation of biological age

Aging is believed to occur across multiple domains, one of which is body composition; however, attempts to integrate it into biological age (BA) have been limited. Here, we consider the sex-dependent role of anthropometry for the prediction of 10-year all-cause mortality using data from 18,794 NHANES participants to generate and validate a new BA metric. Our data-driven approach pointed to sex-specific contributors for BA estimation: WHtR, arm and thigh circumferences for men; weight, WHtR, thigh circumference, subscapular and triceps skinfolds for women. We used these measurements to generate AnthropoAge, which predicted all-cause mortality (AUROC 0.876, 95%CI 0.864–0.887) and cause-specific mortality independently of ethnicity, sex, and comorbidities; AnthropoAge was a better predictor than PhenoAge for cerebrovascular, Alzheimer, and COPD mortality. A metric of age acceleration was also derived and used to assess sexual dimorphisms linked to accelerated aging, where women had an increase in overall body mass plus an important subcutaneous to visceral fat redistribution, and men displayed a marked decrease in fat and muscle mass. Finally, we showed that consideration of multiple BA metrics may identify unique aging trajectories with increased mortality (HR for multidomain acceleration 2.43, 95%CI 2.25–2.62) and comorbidity profiles. A simplified version of AnthropoAge (S-AnthropoAge) was generated using only BMI and WHtR, all results were preserved using this metric. In conclusion, AnthropoAge is a useful proxy of BA that captures cause-specific mortality and sex dimorphisms in body composition, and it could be used for future multidomain assessments of aging to better characterize the heterogeneity of this phenomenon.