ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Differential distribution of aldolase A and C in the human central nervous system

We have analyzed the distribution of aldolase A and C mRNAs and proteins in various areas of the human brain using Northern blot analyses and immunohistochemistry. Aldolase A mRNA expression was higher than aldolase C mRNA expression in all areas of the brain examined. Aldolase C mRNA expression was highest in the cerebellum. Aldolase C protein was present in well-delimited regions of the CNS, and was distributed in stripes in the Purkinje cell layer of the cerebellum, in the inferior olives and in the sensory neurons of the posterior horn of the spinal cord. The novel finding of aldolase C in well-delimited cell compartments of the human cerebellum and in several other areas of the CNS lends weight to the hypothesis that this protein exerts other functions (e.g. sensory transmission) besides those characteristic of a glycolytic enzyme.     

Leptin, Leptin Receptors and ACTH Immunoreactivities are Present in the Gastrointestinal Tract and the Neural Tube of Tadpoles of the Newt Triturus

Leptin, its receptor and ACTH were detected by immunohistochemistry in the gastrointestinal tract and the neural tube of the amphibian urodele, Triturus cristatus carnifex, during development. These molecules were found after hatching of tadpoles, starting from stage 41. In the gastrointestinal tract, cells immunoreactive to leptin and its receptor were first revealed in the stomach, the liver and the gut and then in the pancreas. Both immunoreactives were colocalized in the same cells in some areas. Immunostaining for ACTH appeared at stages 43/45 in the stomach, the gut and the pancreas. In adjacent sections, a few cells immunoreactive to both ACTH and leptin receptor were detected. A few cells were immunoreactive to both insulin and leptin receptor. Immunoreactivities to leptin and its receptor were also found in adjacent sections of the neural tube, often colocalized in the same cell. Moreover, in prosencephalon, mesencephalon, rhomboencephalon and spinal cord, ACTH-immunoreactive cells were detected in the same areas as the leptin receptor immunoreactive cells. These results suggest the existence of a neuroendocrine network in newt tadpoles both at the central level, where it resembles that of mammals, and at the peripheral level, where it may act locally to regulate food intake and metabolism, e.g. yolk digestion.     

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.     

An inquiry-based learning model for an exercise physiology laboratory course

We developed an inquiry-based learning model to better stimulate undergraduate students' cognitive development of exercise physiology laboratory concepts. The course core is the two independent research projects that students, working in small groups, complete during the last 9 wk of the semester. Student groups develop their own research question and hypothesis, design the experiment, collect and analyze the data, and report their findings to the rest of the class using presentation software. To help with success of the research projects, students are taken through a series of guided-inquiry laboratory activities during the initial 6 wk of the semester to develop laboratory skills and an understanding of the scientific process. Observations of student behaviors reflected a high level of enthusiasm and engagement in laboratory activities. Surveys, journal entries, and interviews indicated that students felt empowered by having ownership in their projects, which may be the key reason for the success of this model.     

Models of central pattern generators for quadruped locomotion I. Primary gaits

In this paper we continue the analysis of a network of symmetrically coupled cells modeling central pattern generators for quadruped locomotion proposed by Golubitsky, Stewart, Buono, and Collins. By a cell we mean a system of ordinary differential equations and by a coupled cell system we mean a network of identical cells with coupling terms. We have three main results in this paper. First, we show that the proposed network is the simplest one modeling the common quadruped gaits of walk, trot, and pace. In doing so we prove a general theorem classifying spatio-temporal symmetries of periodic solutions to equivariant systems of differential equations. We also specialize this theorem to coupled cell systems. Second, this paper focuses on primary gaits; that is, gaits that are modeled by output signals from the central pattern generator where each cell emits the same waveform along with exact phase shifts between cells. Our previous work showed that the network is capable of producing six primary gaits. Here, we show that under mild assumptions on the cells and the coupling of the network, primary gaits can be produced from Hopf bifurcation by varying only coupling strengths of the network. Third, we discuss the stability of primary gaits and exhibit these solutions by performing numerical simulations using the dimensionless Morris-Lecar equations for the cell dynamics.     

Models of central pattern generators for quadruped locomotion II. Secondary gaits

We continue the analysis of the network of symmetrically coupled cells modeling central pattern generators (CPG) for quadruped locomotion proposed by Golubitsky, Stewart, Buono and Collins by studying secondary gaits. Secondary gaits are modeled by output signals from the CPG where each cell emits one of two different output signals along with exact phase shifts. Examples of secondary gaits are transverse gallop, rotary gallop, and canter. We classify secondary gaits that bifurcate when the Poincaré map of a primary gait has a real eigenvalue crossing the unit circle. In particular, we show that periodic solutions modeling transverse gallop and rotary gallop bifurcate from primary gaits. Moreover, we find gaits from period-doubling bifurcations and analyze plausible footfall patterns. Numerical simulations are performed using the Morris-Lecar equations as cell dynamics.     

Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pK_as of 14 ionizable residues. An all-atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pK_as in the protein, in contrast to multiple simulations for the free energy perturbation method. pK_int shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson-Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long-range interactions on the pK_int shifts is discussed. © 1994 Wiley-Liss, Inc.