Glycosidases and Cerebellar Ontogenesis in the Rat

Abstract: Five glysosidases (α- and β-D-mannosidase, β-D-galactosidase, β- N -acetyl-glucosaminidase, and α-L-fucosidase) were studied during the postnatal development of the rat cerebellum. Each glycosidase has a particular developmental pattern. Transient decreases in the specific activities of β-mannosidase, β-D-galactosidase, and β-D- N -acetyl-glucosaminidase could be correlated with the phase of massive cell multiplication. A possible more specific role for α-D-mannosidase is discussed.     

Improved fatigue resistance in Gsα-deficient and aging mouse skeletal muscles due to adaptive increases in slow fibers

Genetically modified mice with deficiency of the G protein α-subunit (G(s)α) in skeletal muscle showed metabolic abnormality with reduced glucose tolerance, low muscle mass, and low contractile force, along with a fast-to-slow-fiber-type switch (Chen M, Feng HZ, Gupta D, Kelleher J, Dickerson KE, Wang J, Hunt D, Jou W, Gavrilova O, Jin JP, Weinstein LS. Am J Physiol Cell Physiol 296: C930-C940, 2009). Here we investigated a hypothesis that the switching to more slow fibers is an adaptive response with specific benefit. The results showed that, corresponding to the switch of myosin isoforms, the thin-filament regulatory proteins troponin T and troponin I both switched to their slow isoforms in the atrophic soleus muscle of 3-mo-old G(s)α-deficient mice. This fiber-type switch involving coordinated changes of both thick- and thin-myofilament proteins progressed in the G(s)α-deficient soleus muscles of 18- to 24-mo-old mice, as reflected by the expression of solely slow isoforms of myosin and troponin. Compared with age-matched controls, G(s)α-deficient soleus muscles with higher proportion of slow fibers exhibited slower contractile and relaxation kinetics and lower developed force, but significantly increased resistance to fatigue, followed by a better recovery. G(s)α-deficient soleus muscles of neonatal and 3-wk-old mice did not show the increase in slow fibers. Therefore, the fast-to-slow-fiber-type switch in G(s)α deficiency at older ages was likely an adaptive response. The benefit of higher fatigue resistance in adaption to metabolic deficiency and aging provides a mechanism to sustain skeletal muscle function in diabetic patients and elderly individuals.     

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10‐R98S). Our data support that RPL10‐R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock‐in of RPL10‐R98S was associated with a 1.7‐ to 2.5‐fold increased production of four transiently expressed recombinant proteins and 1.7‐fold for one out of two stably expressed proteins. In CHO‐S cells, eGFP reached a 2‐fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10‐R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10‐R98S phenotypes can maximize its exploitability for the mammalian protein production industry.     

Evolutionary responses of tree phenology to the combined effects of assortative mating,gene flow and divergent selection

The timing of bud burst (TBB) in temperate trees is a key adaptive trait, the expression of which is triggered by temperature gradients across the landscape. TBB is strongly correlated with flowering time and is therefore probably mediated by assortative mating. We derived theoretical predictions and realized numerical simulations of evolutionary changes in TBB in response to divergent selection and gene flow in a metapopulation. We showed that the combination of the environmental gradient of TBB and assortative mating creates contrasting genetic clines, depending on the direction of divergent selection. If divergent selection acts in the same direction as the environmental gradient (cogradient settings), genetic clines are established and inflated by assortative mating. Conversely, under divergent selection of the same strength but acting in the opposite direction (countergradient selection), genetic clines are slightly constrained. We explored the consequences of these dynamics for population maladaptation, by monitoring pollen swamping. Depending on the direction of divergent selection with respect to the environmental gradient, pollen filtering owing to assortative mating either facilitates or impedes adaptation in peripheral populations.     

Galactomannoproteins of Aspergillus fumigatus

Galactofuranose-containing molecules have been repeatedly shown to be important antigens among human fungal pathogens, including Aspergillus fumigatus. Immunogenic galactofuran determinants have been poorly characterized chemically, however. We reported here the characterization of two glycoproteins of A. fumigatus with an N-glycan containing galactofuranose. These proteins are a phospholipase C and a phytase. Chemical characterization of the N-glycan indicates that it is a mixture of Hex(5-13)HexNAc(2) oligosaccharides, the major molecular species corresponding to Hex(6-8)HexNAc(2). The N-glycan contained one galactofuranose unit that was in a terminal nonreducing position attached to the 2 position of Man. This single terminal nonreducing galactofuranose is essential for the immunoreactivity of the N-glycans assessed either with a monoclonal antibody that recognizes a tetra-beta-1,5-galactofuran chain of galactomannan or with Aspergillus-infected patient sera.     

Fine-scale population structure and dispersal in Biomphalaria glabrata, the intermediate snail host of Schistosoma mansoni, in Venezuela

Biomphalaria glabrata is the main intermediate host of Schistosoma mansoni in America and one of the most intensely studied species of freshwater snails, yet very little is known about its population biology. Here, we used seven highly polymorphic microsatellite loci to analyse genetic diversity in the Valencia lake basin, which represents the core of the endemic area for schistosomiasis in Venezuela. Populations were sampled at short spatial scale (a few kilometres), both inside the lake and in ponds or rivers near the lake. Our results indicate that B. glabrata essentially cross-fertilizes, with little variation in selfing rates among populations. Our markers detected considerable genetic variation, with an average heterozygosity of 0.60. More diversity per population was found within than outside the lake, suggesting an influence of connectivity among populations on the levels of genetic diversity. A marked population structure was detected and lake populations were less structured than other populations. Most individuals were assigned to their population of origin using an assignment test. No strong demographic signal (e.g. bottleneck) was detected, though lake populations are likely to experience bottlenecks more frequently than the other populations analysed. Differences in gene flow therefore seem to play an important role in population differentiation and in the restoring of genetic diversity in demographically unstable populations.     

Degradation of polycyclic aromatic hydrocarbons by pure strains and by defined strain associations: inhibition phenomena and cometabolism

Six bacterial strains capable of using, as sole carbon and energy source, at least one of the following polycyclic aromatic hydrocarbons (PAH), naphthalene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene, were isolated. The interactions between these PAH during their biodegradation were studied in experiments involving PAH pairs, one PAH at least being used as a carbon source. All individual strains were found capable of cometabolic degradation of PAH in a range varying among strains. Inhibition phenomena, sometimes drastic, were often observed but synergistic interactions were also detected. Naphthalene was toxic to all strains not isolated on this compound. Strain associations were found efficient in relieving inhibition phenomena, including the toxic effect of naphthalene. Accumulation of water-soluble metabolites was consistently observed during PAH degradation.     

Cloning and Nucleotide Sequence of the Endoinulinase-Encoding Gene, inu2, from Aspergillus ficuum

A 2.3 kb DNA fragment that contains a gene encoding endoinulinase, inu2, from Aspergillus ficuum ATCC 16882 was isolated and analyzed. It includes an open reading frame of 1,551 bp, coding for a polypeptide with calculated molecular weight of 55,790 Da, including a putative signal peptide of 22 amino acids. Alignment of amino acid sequences revealed 73.3% identity and 93.9% similarity between A. ficuum and Penicillium purpurogenum endoinulinase.