Localized decrease of beta-catenin contributes to the differentiation of human embryonic stem cells

Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies.     

Periodic breathing in the mouse.

The hypothesis was that unstable breathing might be triggered by a brief hypoxia challenge in C57BL/6J (B6) mice, which in contrast to A/J mice are known not to exhibit short-term potentiation; as a consequence, instability of ventilatory behavior could be inherited through genetic mechanisms. Recordings of ventilatory behavior by the plethsmography method were made when unanesthetized B6 or A/J animals were reoxygenated with 100% O(2) or air after exposure to 8% O(2) or 3% CO(2)-10% O(2) gas mixtures. Second, we examined the ventilatory behavior after termination of poikilocapnic hypoxia stimuli in recombinant inbred strains derived from B6 and A/J animals. Periodic breathing (PB) was defined as clustered breathing with either waxing and waning of ventilation or recurrent end-expiratory pauses (apnea) of > or = 2 average breath durations, each pattern being repeated with a cycle number > or = 3. With the abrupt return to room air from 8% O(2), 100% of the 10 B6 mice exhibited PB. Among them, five showed breathing oscillations with apnea, but none of the 10 A/J mice exhibited cyclic oscillations of breathing. When the animals were reoxygenated after 3% CO(2)-10% O(2) challenge, no PB was observed in A/J mice, whereas conditions still induced PB in B6 mice. (During 100% O(2) reoxygenation, all 10 B6 mice had PB with apnea.) Expression of PB occurred in some but not all recombinant mice and was not associated with the pattern of breathing at rest. We conclude that differences in expression of PB between these strains indicate that genetic influences strongly affect the stability of ventilation in the mouse.     

Decomposition patterns of unprocessed and processed lignocellulosics in a freshwater fish pond

In an attempt to recycle the lignocellulosic wastes like Eichhornia crassipes, Salvinia cucullata and rice (Oryza sativa) straw as manurial inputs in freshwater fish pond ecosystem, a decomposition experiment was carried out in litter bags in an oligotrophic freshwater fish pond environment, with the above mentioned three substrates in unprocessed and microbially processed forms. The loss rates, associated microbial groups, oxygen consumption patterns and other related parameters like carbon, nitrogen, phosphorus, cellulose, hemicellulose and lignin were analysed. The mean daily dry matter loss rates (unprocessed: 10.44>6.97>1.97 and processed: 11.03>8.21>3.67) and oxygen uptake rate (unprocessed: 0.675>0.571>0.568 mg O₂ g^–1 h^–1 and processed: 0.592>0.424>0.407 mg O₂ g^–1 h^–1) in raw and processed substrates were in the sequence Eichhornia > rice straw > Salvinia. The oxygen consumption pattern almost covariated with variations in temperature of pond water, daily dry matter loss rates and fungal counts on substrates. During the decay, the percentage of N and P increased whereas that of C decreased, resulting in lowering of C/N and C/P ratios of the substrates. The structural polymeric fractions like cellulose and hemicellulose decreased along with dry matter whereas the lignin content increased after an initial decrease due to loss of other structural carbohydrates resulting in apparent per cent gain of lignin. A higher number of different heterotrophic bacterial groups was observed in the processed substrates as compared to their raw counterparts. However, cellulolytic bacterial numbers were found to fluctuate through the study period. The fungal load was found to be decreasing gradually as the decay progressed. In this study, bacteria were found to be the prominent microbial group responsible for the decay. The nitrogen-fixing, phosphatase-producing and phosphorus-solubilising bacterial groups were observed to play an important role in lowering the C/N and C/P ratios of the decomposing substrates during decay.     

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

Investigations on the artificial synthesis of amphidiploids of Brassica tournefortii Gouan with the other elementary species of Brassica. I. Genomic relationships

With the object of studying the genomic relationships of Brassica tournefortii Gouan with the other elementary species of Brassica viz. B. campestris (2n=20, A genome), B. oleracea (2n=18, C genome) and B. nigra (2n=16, B genome), it has been hybridized with them. The percentage of F₁ hybrids formed, their morphology and meiotic behaviour have been described. Based upon crossability relationships and meiotic pairing in the F₁ hybrids, it is inferred that the D genome of B. tournefortii is more closely related to the A genome than to the B and C genomes. It may have been derived from the A genome which likewise shows a strong genetic isolation from B and C. The species has developed a strong genetic barrier in the course of its evolution and shows little crossability, high hybrid sterility and no gene flow with any of the other elementary species. The fact that it has not formed any natural amphidiploids with the elementary species which otherwise are formed in all combinations, is more evidence that it originated more recently than the A genome. It is presumed that B. tournefortii, being more distantly related to B. nigra than to other elementary species, may form stable artificial aphid resistant amphidiploids with the former.     

Role of 677C→T polymorphism a single substitution in methylenetetrahydrofolate reductase (MTHFR) gene in North Indian infertile men

Failure or severe difficulty in conceiving a child is surprisingly common, worldwide problem. Half of these cases are due to male factors with defects in sperm (1 in 15 men) being the single most common cause. Also about 60–75 % of male infertility cases are idiopathic, since the molecular mechanisms underlying the defects remain unknown. DNA methylation is crucial for spermatogenesis and high methylenetetrahydrofolate reductase (MTHFR) activity in adult testis than other organs in mouse, signifies its critical role in spermatogenesis. According to recent findings there is a correlation of epigenetic regulation of several imprinted genes with disturbed spermatogenesis and fertility. Consequently any change in the MTHFR gene sequence can modify the spermatogenesis including transmission of infertility to the carriers. The aim of the study is to analyze the distribution of the single nucleotide polymorphism C677T in the MTHFR gene in 637 North Indian infertile patients and 364 fertile North Indian men as controls by using PCR–RFLP technique and Chi Square test for statistical analysis. The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total infertile men were 70.17, 24.17, 5.65 % in infertile men and 75.27, 21.7, 2.74 % in controls, respectively. The average frequency of the MTHFR 677T allele was 17.73 % in infertile men as compared to 13.59 % in controls. The statistical difference was significant. Disease risk was found 2.27-folds increased in patients who were carrying T allele. We found an association of C677T polymorphism with male infertility and that it may be a genetic risk factor for male infertility in North Indian population.