A novel Golgi mannosidase inhibitor: Molecular design,synthesis, enzyme inhibition,and inhibition of spheroid formation

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell–cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.     

Urbanization effects on different biological organization levels of an estuarine polychaete tolerant to pollution

Estuarine species exposed to diffuse contaminants might trigger either positive or negative feedbacks in many biological scales. Their life history traits performing at different biological organization levels could propose an organism as a useful indicator of environmental pollution, mainly addressed as sensitive or tolerant species. To track the effects of contaminants from the molecular to the population level of the polychaete Laeonereis acuta we utilize a framework of biomarkers. For this purpose we assessed the L. acuta frequency of micronuclei at the molecular level, the body size and biomass at individual level, and the production-to-biomass ratio at population level in five urbanized and five non-urbanized estuaries in southern Brazil. L. acuta had significantly varying positive and negative feedbacks between urbanized and non-urbanized estuaries at multiple biological scales. These generalized effects in all biological organization scales indicate a pollution impact on the polychaete. The main responses accounted for individuals becoming lengthy and weighty, but with molecular damage. The L. acuta allocation of energy to body enlargement in polluted environments, and a consequent reduced population turnover, contradicts the expected from an opportunist species. The damages in DNA and the internal strategies of individuals, as antioxidant defense mechanisms, could favor resistance of the population and tolerance to pollutants. All of these characteristics induce bioaccumulation and could cause bottom-up pollution transfer compromising the estuarine food web. These results, ascertain that L. acuta could be considered as a tolerant species, instead of an opportunistic, and as a useful indicator of environmental pollution in estuaries.     

The effects of maturation on self-induced dynamic body sway frequencies of girls performing acrobatics or classical dance

We investigated the effects of maturation on the dynamic body sways of healthy girls. Prepubertal and postpubertal girls practising professional physical activities requiring a good ability to maintain equilibrium (acrobats and dancers) were asked to stand on a free seesaw platform and the results compared to those for untrained age-matched girls. This platform (stabilometer) allows self-induced body sways. Stabilograms were obtained by a double integration of the angular acceleration from the recordings of the platform sways made with an accelerometer. Fast Fourier transform processing of stabilograms allowed spectral frequency analysis. The total spectrum energy and the energies of three frequency bands (0–0.5 Hz, 0.5–2 Hz, 2–20 Hz) were determined. ANOVA showed that, for all groups of different equilibrium activity and independent of visual input, prepubertal girls had higher energy values than postpubertal girls in the 0- to 0.5-Hz band whereas the opposite was true for 0.5- to 2-Hz band. Ballet dancers were more dependent than acrobats on visual inputs for the regulation of their postural control but were less dependent than untrained girls at both ages. Maturation seemed to shift body sways towards higher frequencies and the utilization of the cues of postural control was different according to the type of equilibrium activity practised by the subjects. Accepted: 7 February 1997     

The energetics of middle-distance running

In order to assess the relative contribution of aerobic processes to running velocity (v), 27 male athletes were selected on the basis of their middle-distance performances over 800, 1500, 3000 or 5000 m, during the 1987 track season. To be selected for study, the average running velocity (v) corresponding to their performances had to be superior to 90% of the best French v of the season. Maximum O2 consumption (VO2max) and energy cost of running (C) had been measured within the 2 months preceding the track season, which, together with oxygen consumption at rest (VO2rest) allowed us to calculate the maximal v that could be sustained under aerobic conditions: vamax = (VO2max - VO2rest) x C-1. The treadmill running v corresponding to a blood lactate of 4 mmol.l-1 (vla4), was also calculated. In the whole group, C was significantly related to height (r = -0.43; P less than 0.03). Neither C nor VO2max (with, in this case, the exception of the 3000 m athletes) were correlated to v. On the other hand, vamax was significantly correlated to v over distances longer than 800 m. These v were also correlated to vla4. However vla4 occurred at 87.5% SD 3.3% of vamax, this relationship was interpreted as being an expression of the correlation between vamax and v. Calculation of vamax provided a useful means of analysing the performances. At the level of achievement studied, v sustained over 3000 m corresponded to vamax.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Optimized Proteomics Approach Reveals Novel Alternative Proteins in Mouse Liver Development

Alternative ORFs (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry as the only direct way of identification is hampered by the short lengths and relative low abundance of AltProts. There is an urgent need for improvement of mass spectrometry methodologies for AltProt identification. Here, we report an approach based on size-exclusion chromatography for simultaneous enrichment and fractionation of AltProts from complex proteome. This method greatly simplifies the variance of AltProts discovery by enriching small proteins smaller than 40 kDa. In a systematic comparison between 10 methods, the approach we reported enabled the discovery of more AltProts with overall higher intensities, with less cost of time and effort compared to other workflows. We applied this approach to identify 89 novel AltProts from mouse liver, 39 of which were differentially expressed between embryonic and adult mice. During embryonic development, the upregulated AltProts were mainly involved in biological pathways on RNA splicing and processing, whereas the AltProts involved in metabolisms were more active in adult livers. Our study not only provides an effective approach for identifying AltProts but also novel AltProts that are potentially important in developmental biology.     

A cladistic and comparative analysis of kinematic components of the fast-start of fishes,with a note on body size constraints

The fast-start is an ecologically relevant behavior pattern in fishes. The present article analyses the distribution of five continuous kinematic traits (latency for response initiation, time to maximum angular velocity, time to maximum displacement velocity, maximum angular velocity, and maximum displacement velocity) in eight of the eleven species described in Eaton (66:65–81, 1977). Phylogenetic generalized least square estimation of ancestor states demonstrated evolutionary changes in maximum angular velocity and maximum displacement velocity, consistent with species differences in the same variables. These changes in maximum velocity are also correlated (phylogenetically independent contrasts) with the mean body sizes of all species, pointing to the possibility that body size was an evolutionary constraint on maximum velocities. The conservation of the other traits suggest that they are mainly constrained by neural control, and a trade-off between neural and body size-constraints is proposed ex hypothesi. An erratum to this article can be found at     

Enteric and serological distribution of serotonin and its precursor tryptophan in perinatal low and normal weight piglets

Perinatal mortality is high among small-for-gestational age (SGA) piglets and continues to be an economic burden and threat to animal welfare. As the physiological role of serotonin (5-hydroxytryptamine, 5-HT) in perinatal development and gastrointestinal function in the pig remains unknown, the aim of this study was to assess the enteric distribution of 5-HT cells and to determine 5-HT together with its precursor tryptophan in the serum of perinatal normal and SGA piglets. For this purpose, proximal and distal parts of the small intestine (SI) were processed for immunohistochemical analysis to assess the presence of 5-HT endocrine cells. Serum 5-HT was measured with ELISA, whereas its precursor, that is, the free fraction of tryptophan (FFT) together with albumin-bound tryptophan and total tryptophan, were analysed with HPLC in postnatal piglets. In addition, the morphological growth patterns of the different intestinal tissue layers of both normal and SGA piglets were stereologically analysed. The stereological volume density of 5-HT enteroendocrine cells showed a significant interaction effect between age and region. Indeed, the amount of 5-HT cells in both the proximal and distal part of the SI tended to decrease according to age, with the lowest values detected at day 3 postpartum. No differences could be observed related to BW. Interestingly, the serum concentration of 5-HT was higher in normal piglets compared with SGA piglets. Moreover, the ratio of FFT to total tryptophan was significantly affected by age and BW. Normal piglets had, on average, a lower FFT/total tryptophan ratio compared with SGA piglets. An approximate linear decrease was observed with increasing age. Finally, the immaturity of the intestinal system of the SGA piglets was not reflected in altered volume densities of the different intestinal layers. To conclude, although no BW effect could be detected in the distribution of enteric 5-HT cells, serum 5-HT and the ratio of FFT to total tryptophan ratio showed significant differences between normal piglets and their SGA littermates.     

Cytokeratin immunoreactivity in lobular intraepithelial neoplasia.

Eighteen commercially available antibodies reactive against different cytokeratin proteins were tested on classic examples of lobular intraepithelial neoplasia (LIN) and of ductal intraepithelial neoplasia (DIN) of the breast. About 90% of higher-grade DIN (AIDH and DCIS) show no or substantially diminished reaction with clone 34betaE12 (specified as reactive against keratins 1, 5, 10, and 14 as determined by the manufacturer), while the cells of LIN were found to express the antigen reactive with this antibody. To determine which of these four keratins are present in the cells of LIN, antibodies reactive against these individual four keratins were tested. None of the four antibodies to keratins 1, 5, 10, or 14 reacted with the cells of LIN. To investigate this further, 13 additional monoclonal antibodies to various other keratin proteins were tested on the cells of LIN. Those that successfully reacted with the cells of LIN were further tested on the cells of DIN. All of the individual antibodies reactive with the cells of LIN were also reactive with the cells of DIN to a degree, with clone RCK108 (reactive against keratin 19) coming the closest to demonstrating the reactivity seen with 34betaE12. We conclude that the reactivity seen in the cells of LIN with 34betaE12 is due to either (a) a crossreaction with keratin 19 that is slightly less prominent than the reaction of the individual clone RCK108, (b) a crossreaction with a keratin protein that was not tested (3, 11, 12), (c) a crossreaction with a protein closely resembling keratin in formalin-fixed, paraffin-embedded tissue, or (d) the detection of a mutated or truncated form of keratin 1, 5, 10, or 14 that cannot be detected by the individual monoclonal antibody.