Lysosomal enzyme oligosaccharide phosphorylation in mouse lymphoma cells: specificity and kinetics of binding to the mannose 6-phosphate receptor in vivo

Phosphomannosyl residues on lysosomal enzymes serve as an essential component of the recognition marker necessary for binding to the mannose 6-phosphate (Man 6-P) receptor and translocation to lysosomes. The high mannose-type oligosaccharide units of lysosomal enzymes are phosphorylated by the following mechanism: N-acetylglucosamine 1-phosphate is transferred to the 6 position of a mannose residue to form a phosphodiester; then N- acetylglucosamine is removed to expose a phosphomonoester. We examined the kinetics of this phosphorylation pathway in the murine lymphoma BW5147.3 cell line to determine the state of oligosaccharide phosphorylation at the time the newly synthesized lysosomal enzymes bind to the receptor. Cells were labeled with [2-(3)H]mannose for 20 min and then chased for various times up to 4 h. The binding of newly synthesized glycoproteins to the Man 6-P receptor was followed by eluting the bound ligand with Man 6-P. Receptor-bound material was first detected at 30 min of chase and reached a maximum at 60 min of chase, at which time approximately 10 percent of the total phosphorylated oligosaccharides were associated with the receptor. During longer chase times, the total quantity of cellular phosphorylated oligosaccharides decreased with a half-time of 1.4 h, suggesting that the lysosomal enzymes had reached their destination and had been dephosphorylated. The structures of the phosphorylated aligosaccharides of the eluted ligand were then determined and compared with the phosphorylated oligosaccharides of molecules which were not bond to the receptor. The major phosphorylated oligosaccharide species present in the nonreceptor-bound material contained a single phosphosphodiester at all time examined. In contrast, receptor-bound oligosaccharides were greatly enriched in species possessing one and two phosphomonoesters. These results indicate that binding of newly synthesized lysosomal enzymes to the Man 6-P receptor occurs only after removal of the covering N- acetylglucosamine residues.     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

TGF-beta expression during rat pregnancy and activity on decidual cell survival

Background  

During early rat pregnancy, trophoblast of the tiny embryo joins with the endometrium and epithelial cells undergo apoptosis. Near the end of pregnancy, regression of the decidua basalis (DB) is also observed (from day 14 to 20). However, little is known about the intra-cellular and molecular mechanisms involved in apoptosis regulation in the uterus during pregnancy. The objective of the present study was to investigate the presence and the developmental expression of transforming growth factor-beta isoforms (TGF-beta well known differentiation factor) in the rat endometrium throughout pregnancy and its action in vitro using cultured endometrial stromal cells.     

Genotype identity has a more important influence than genotype diversity on shoot biomass productivity in willow short‐rotation coppices

Willow (Salix spp.) short‐rotation coppice is commercially grown to produce lignocellulosic biomass to meet renewable bioenergy demands. Most commercial willow coppices are grown in stands of a single genotype, but biomass productivity may be greater in mixed communities, and the productivity in mixed communities may depend on the specific genotypes involved. We assessed the biomass production of four different Salix genotypes (“Björn,” “Jorr,” “Loden,” “Tora”) grown without additional nutrient fertilization during one cutting cycle at three locations in Europe (Uppsala in Sweden, Rostock and Freiburg in Germany) in plots of pure and mixed communities. We evaluated (i) the effect of genotype diversity on shoot biomass productivity, including the evidence for complementarity and selection effects; (ii) the influence of individual genotypes on mixed community productivity; and (iii) the productivity of individual genotypes in response to pure vs. mixed culture. Mean shoot biomass production after the first cutting cycle decreased in the order Rostock (8.7 Mg ha^?1) > Freiburg (6.9 Mg ha^?1) > Uppsala (5.7 Mg ha^?1), with values similar to those for other nonfertilized willow stands after the first growth cycle. Consistently across all three locations, increasing genotype diversity did not significantly affect shoot biomass production. Using Bayesian statistics, the addition of the genotypes “Jorr” and “Loden” was predicted to enhance shoot biomass production, while “Tora” and “Björn” are more likely to reduce shoot biomass production in mixed communities. In addition, we found evidence for a negative selection effect due to the genotype “Tora” performing better in mixed than in pure communities in two of the sites (Freiburg, Uppsala). In conclusion, our results imply that increasing genetic richness has no negative effect on productivity and that there is a potential to design site‐specific genotype mixtures of short‐rotation coppice promoting both high genetic diversity and high biomass production.     

单链胰岛素前体在酵母中的分泌表达及其转变成人胰岛素

将化学合成的单链猪胰岛素前体(PIP)基因和用聚合酶链反应得到的α交配因子前导顺序(αMFL)基因插入质粒pVT102-U的醇脱氢酶基因ADH1的启动子和3’终止顺序之间而生成质粒pVT102-U/αMFL-PIP.被pVT102-U/αMFL-PIP转化的酵母(Saccharomyces cerevistae)可表达单链前体并分泌到培养基中.前体在纯化后可通过胰蛋白酶的转肽作用转变成人胰岛素.纯化的人胰岛素具有全部活力并可结晶.人胰岛素的总收率为每升培养液25mg.     

Molecular phylogeny of the springhare, Pedetes capensis, based on mitochondrial DNA sequences

The phylogenetic position of the Pedetidae, represented by a single species Pedetes capensis, is controversial, reflecting in part the retention of both Hystricomorphous and Sciurognathous characteristics in this rodent. In an attempt to clarify the species evolutionary relationships, mtDNA gene sequences from 10 rodent species (representing seven families) were analyzed using phenetic, parsimony, and maximum-likelihood methods of phylogenetic inference; the rabbit, Oryctolagus cuniculus (Order Lagomorpha), and cow, Bos taurus (Order Artiodactyla), were used as outgroups. Investigation of 714 base pairs of the protein-coding cytochrome b gene indicate strong base bias at the third codon position with significant rate heterogeneity evident between the three structural domains of this gene. Similar analyses conducted on 816 base pairs of the 12S rRNA gene revealed a transversion bias in the loop sections of all taxa. The cytochrome b gene sequences proved useful in resolving associations between closely related species but failed to produce consistent tree topologies at the family level. In contrast, phylogenetic analysis of the 12S rRNA gene resulted in strong support for the clustering of Pedetidae/Heteromyidae/Geomyidae and Muridae in one clade to the exclusion of the Hystricidae/Thryonomyidae and Sciuridae, a finding which is concordant with studies of rodent fetal membranes as well as reproductive and other anatomical features.      

The C-S Lyases of Higher Plants : Direct Comparison of the Physical Properties of Homogeneous Alliin Lyase of Garlic (Allium sativum) and Onion (Allium cepa)

Garlic and onion alliin lyases, although from closely related species, have many differences. The two enzymes differ in their K_m values, pH optima, and isoelectric points. There is a major difference in their molecular weight and subunit structure. The garlic holoenzyme has a molecular weight of 85,000 and consists of two subunits of molecular weight 42,000. The onion enzyme has a holoenzyme molecular weight of 200,000 composed of four subunits of molecular weight 50,000. The onion enzyme is much more difficult to dissociate into its subunits which suggests differences in subunit interaction between the two enzymes. The dimeric stucture of the garlic and the tetrameric structure of the onion enzyme is consistent with a coenzyme content (pyridoxal-5′-phosphate) equivalent to one mole per subunit. The two enzymes vary vastly in their spectra, the onion enzyme having a lower pyridoxal-5′-phosphate absorbance at 430 nanomoles and an inability to react with l-cysteine. Both enzymes are glycoproteins and bind to concanavalin A-Sepharose columns. The onion alliin lyase binds more tightly than the garlic enzyme. The amino acid content of both enzymes is similar as is the carbohydrate content. However, upon hydrolysis the onion lyase does yield more mannose units than the garlic enzyme which is consistent with the former's stronger affinity for concanavalin A.     

Morphine suppresses the ovarian steroid hormone-dependent lordosis response of female guinea pigs: reversal by naloxone but not clonidine.

The opiate agonist morphine caused a dose- and time-dependent suppression of lordosis responding in ovariectomized guinea pigs treated with estradiol-17 beta and progesterone. The suppression of lordosis by morphine appears to be mediated by opiate receptors since the opiate antagonist naloxone blocked its effects both in terms of the percentage of animals showing lordosis and the duration of individual responses. Naloxone, when given alone, did not affect lordosis responding in estradiol-17 beta + progesterone-primed animals and did not induce lordosis in animals primed with estradiol-17 beta alone. Thus, endogenous opioids might not tonically inhibit lordosis under the physiological conditions examined. The alpha-noradrenergic agonist clonidine did not reverse the effects of morphine on lordosis. Thus, the inhibitory effects of morphine on this behavior might be independent of its presynaptic effects on norepinephrine release in brain.