Genetic factors contributing to defective spermatogonial differentiation in juvenile spermatogonial depletion (Utp14bjsd) mice

Male mice that are homozygous for the juvenile spermatogonial depletion (jsd) mutation in the Utp14b gene undergo several waves of spermatogenesis. However, spermatogonial differentiation ceases and in adults, spermatogonia are the only germ cells that remain. To understand further the blockage in spermatogonial differentiation in Utp14b(jsd) mutant mice, we correlated the rate and severity of spermatogonial depletion and the restoration of spermatogenesis following the suppression of testosterone or elevation of testicular temperature with the genetic background. Testes from Utp14b(jsd) mutant mice on B6, C3H, and mixed C3H-B6-129 (HB129) genetic backgrounds all showed steady decreases in the numbers of normal spermatogonia between 8 wk and 20 wk of age. The percentages of tubules with differentiating germ cells were higher and the spermatogonia were more advanced in C3H- background than in B6- or HB129-background Utp14b(jsd) mice. Genetic crosses showed that the source of the Y chromosome was a major factor in determining the severity of spermatogonial depletion in Utp14b(jsd) mutant mice. When Utp14b(jsd) mutants were subjected to total androgen ablation or unilateral cryptorchidization, spermatogenic development recovered markedly in the C3H and HB129 background but showed less recovery in the B6-background mice. The differences noted between the strains in terms of the severity of spermatogonial depletion were not dependent upon testosterone level or scrotal temperature but correlated with the magnitudes of the effects of elevated temperature on normal and Utp14b(jsd) mutant spermatogenic cells. Thus, the abilities of germ cells in certain strains to survive elevated temperatures may be related to their abilities to maintain some degree of differentiation potential after the Utp14b(jsd) gene is mutated.     

Dictyostelium gnt15 encodes a protein with similarity to LARGE and plays an essential role in development

LARGE is a putative glycosyltransferase found to be mutated in mice with myodystrophy or patients with congenital muscular dystrophy. By homology searches, we identified in the Dictyostelium discoideum genome four open reading frames, i.e. gnt12-15, encoding proteins with sequence similarity to LARGE. Semi-quantitative RT-PCR analysis revealed distinct temporal expression patterns of the four gnt genes throughout Dictyostelium development. To explore the gene function, we performed targeted disruptions of gnt14 and gnt15. The gnt14(-) strains showed no obvious phenotypes. However, gnt15(-) cells grew slowly, changed in morphology, and displayed a developmental phenotype arresting at early stages. Compared with the wild type, gnt15(-) cells were more adhesive and exhibited altered levels of some surface adhesion molecules. Moreover, lectin-binding analysis demonstrated that gnt15 disruption affected profiles of membrane glycoproteins. Taken together, our data suggest that Gnt15 is essential for Dictyostelium development and may have a role in modulating cell adhesion and glycosylation.     

Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

Extremely Low Frequency Magnetic Fields (ELF MF) has been considered as a “possible human carcinogen” by International Agency for Research on Cancer (IARC) while credible mechanisms of its carcinogenicity remain unknown. In this study, a proteomics approach was employed to investigate the changes of protein expression profile induced by ELF MF in human breast cancer cell line MCF7, in order to determine ELF MF-responsive proteins. MCF7 cells were exposed to 50 Hz, 0.4 mT ELF MF for 24 h and the changes of protein profile were examined using two dimensional electrophoresis. Up to 6 spots have been statistically significantly altered (their expression levels were changed at least 5 fold up or down) compared with sham-exposed group. 19 ones were only detected in exposure group while 19 ones were missing. Three proteins were identified by LC-IT Tandem MS as RNA binding protein regulatory subunit Proteasome subunit beta type 7 precursor and Translationally Controlled Tumor Protein. Our finding showed that 50 Hz, 0.4 mT ELF MF alternates the protein profile of MCF7 cell and may affect many physiological functions of normal cell and 2-DE coupled with MS is a promising approach to elucidating cellular effects of electromagnetic fields.     

Main-chain conformational tendencies of amino acids

A Ramachandran plot is a visual representation of the main-chain conformational tendencies of an amino acid. Despite forty years of research, the shape of Ramachandran plots is still a matter of debate. The issue in making a Ramachandran plot based on experimental data is deciding whether sparse data represent genuine conformations. We present here a simple solution to settle the ambiguities of the sparse data, and explain how we verified the accuracies of our plots using an independent dataset. To obtain our results, we then measured the pair-wise distances of main-chain conformational tendencies among amino acids, and showed that the conformational relationships of amino acids are well preserved in a two-dimensional map, leading to the conclusion that the conformational diversity space of amino acids is largely two dimensional. We further noticed that amino acids in early and late evolutionary stages are located in different zones in the two-dimensional map. In addition to these conclusions, we here present an amino acid substitution table derived from experimental data.     

Protein-Protein Docking Benchmark 2.0: an update

We present a new version of the Protein-Protein Docking Benchmark, reconstructed from the bottom up to include more complexes, particularly focusing on more unbound-unbound test cases. SCOP (Structural Classification of Proteins) was used to assess redundancy between the complexes in this version. The new benchmark consists of 72 unbound-unbound cases, with 52 rigid-body cases, 13 medium-difficulty cases, and 7 high-difficulty cases with substantial conformational change. In addition, we retained 12 antibody-antigen test cases with the antibody structure in the bound form. The new benchmark provides a platform for evaluating the progress of docking methods on a wide variety of targets. The new version of the benchmark is available to the public at http://zlab.bu.edu/benchmark2.     

Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was -6.8 mV (apical side), transepithelial resistance was 1,050 Omega.cm(2), and short-circuit current (I(sc)) was 8.1 microA/cm(2). Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kOmega.cm(2), respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of alpha-epithelial Na(+) channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na(+)-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na(+) flux (19 nmol.min(-1).cm(-2)) was two times as large as the reverse flux, and net transepithelial Na(+) flux was nearly double the amiloride-sensitive I(sc). In plain Ringer solution, the amiloride-sensitive I(sc) went toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl(-)-dependent I(sc) consistent with the stimulation of transepithelial Cl(-) secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na(+) absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl(-) secretion activated by cAMP.     

Spatial and temporal distribution of induced resistance to greenbug (Homoptera: Aphididae) herbivory in preconditioned resistant and susceptible near isogenic plants of wheat

Interactions between biotype E greenbugs, Schizaphis graminum (Rodani), and two near isogenic lines of the greenbug resistance gene Gb3 of wheat, Triticum aestivum L., were examined for 62 d after infestation. By comparing aphid performance and host responses on control and greenbug-preconditioned plants, we demonstrated that systemic resistance to greenbug herbivory was inducible in the resistant genotype with varying intensities and effectiveness in different parts of the plants. Preconditioning of susceptible plants resulted in modification of within-plant aphid distribution and reduction of cumulative greenbug densities, but it showed no effect on reducing greenbug feeding damage to host plant. Preconditioning of resistant plants altered greenbug population dynamics by reducing the size and buffering the fluctuation of the aphid population. Preconditioning in the first (oldest) leaf of the resistant plant had no phenotypically detectable effect in the stem and induced susceptibility locally in the first leaf within the first 2 d after infestation. The preconditioning-induced resistance reduced greenbug density, delayed aphid density peaks and extended the life of younger leaves in resistant plants. Expression of induced resistance was spatially and temporally dynamic within the plant, which occurred more rapidly, was longer in duration, and stronger in intensity in younger leaves. Host resistance gene-mediated induced resistance was effective in lowering greenbug performance and reducing damage from greenbug herbivory in host plants. Results from this study supported the optimal defense theory regarding within-plant defense allocation.     

Anti-inflammatory and antiviral effects of Glossogyne tenuifolia

Glossogyne tenuifolia (Hsiang-Ju) is a traditional antipyretic and hepatoprotective herb used in Chinese medicine. The aim of this research is to investigate the pharmacological activities and potent components of the ethanol extract of Glossogyne tenuifolia (GT) in human primary cells and cell line. We found that GT (0.1 approximately 0.25 mg/ml) exerted dose-dependent inhibitions on the release of TNF-alpha and IL-6 in LPS-activated human whole blood and peripheral blood mononuclear cells (PBMC), and IFN-gamma in PHA-stimulated human whole blood. The lack of cytotoxicity indicated that the inhibitory effects of GT on cytokine production were not due to cell death. Luteolin, the deglycosylated derivative of one of the major compositions, luteolin-7-glucoside, exerted inhibitory effects on TNF-alpha, IL-6 and IFN-gamma production in activated human whole blood with estimated IC(50)s of 42.73 microM, 44.86 microM and 3.34 microM, respectively. Furthermore, GT had potent anti-hepatitis B virus (HBV) effects on the human hepatocellular carcinoma cell line, PLC/PRF/5. GT exhibited a dose-dependent inhibition on the release of hepatitis B surface antigen (HBsAg) by repressing the expression of HBsAg with IC(50) of 0.093 mg/ml. We concluded that GT exerted combinatorial anti-inflammatory and antiviral effects, and the multiple actions may underlie its traditional hepatoprotective function.