de novo Design and Synthesis of Candida antarctica Lipase B Gene and α-Factor Leads to High-Level Expression in Pichia pastoris

Candida antarctica lipase B (CALB) is one of the most widely used and studied enzymes in the world. In order to achieve the high-level expression of CALB in Pichia, we optimized the codons of CALB gene and α-factor by using a de novo design and synthesis strategy. Through comparative analysis of a series of recombinants with different expression components, we found that the methanol-inducible expression recombinant carrying the codon-optimized α-factor and mature CALB gene (pPIC9KαM-CalBM) has the highest lipase production capacity. After fermentation parameters optimization, the lipase activity and protein content of the recombinant pPIC9KαM-CalBM reached 6,100 U/mL and 3.0 g/L, respectively, in a 5-L fermentor. We believe this strategy could be of special interest due to its capacity to improve the expression level of target gene, and the Pichia transformants carrying the codon-optimized gene had great potential for the industrial-scale production of CALB lipase.     

Structure and Expression of a Dhurrinase (β-Glucosidase) from Sorghum

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.     

Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17 β estradiol

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.     

Acquired Antibiotic Resistance: Are We Born with It?

The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including tet(M), ermB, sul2, and bla(TEM) were detected in infant subjects. Enterococcus spp., Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Escherichia coli/Shigella spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure.     

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [¹⁴C]gibberellin (GA)₉ and [¹⁴C]GA₂₀ to radiolabeled GA₄ and GA₁, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA₉ was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA₂₀. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA₁₂-aldehyde, GA₁₂, GA₁₉, GA₂₅, GA₅₃, or GA₄₄ as the open lactone (20-hydroxy-GA₅₃), whereas GA₁₅, GA₂₄, and GA₄₄ were hydroxylated to GA₃₇, GA₃₆, and GA₃₈, respectively. The open lactone of GA₁₅ (20-hydroxy-GA₁₂) was hydroxylated but less efficiently than GA₁₅. In contrast to the free acid, GA₂₅ 19,20-anhydride was 3β-hydroxylated to give GA₁₃. 2,3-Didehydro-GA₉ and GA₅ were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA₉ and GA₆.Dwarf mutants with reduced biosynthesis of the GA plant hormones have been valuable tools in studies of the function of these compounds (Ross, 1994). In Arabidopsis thaliana, mutations at six loci (GA1-GA6) that result in reduced GA biosynthesis have been identified (Koorneef and van der Veen, 1980; Sponsel et al., 1997), and three of these loci have recently been cloned. The GA1 locus was isolated by genomic subtraction (Sun et al., 1992) and shown by heterologous expression in Escherichia coli to encode the enzyme that cyclizes geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya, 1994). This enzyme was formerly referred to as ent-kaurene synthase A but has been renamed copalyl diphosphate synthase (Hedden and Kamiya, 1997; MacMillan, 1997). The GA5 locus was shown to correspond to one of the GA 20-oxidase genes (Xu et al., 1995), the products of which catalyze the conversion of GA₁₂ to GA₉ and GA₅₃ to GA₂₀ (Phillips et al., 1995; Xu et al., 1995). GA 20-oxidases are 2-oxoglutarate-dependent dioxygenases that are encoded by small multigene families, members of which are differentially expressed in plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the basis of the derived amino acid sequence, was also shown to encode a dioxygenase (Chiang et al., 1995). Several lines of evidence indicate that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a ga4 mutant, all alleles of which are semidwarf, contained reduced concentrations of the 3β-hydroxy GAs GA₁, GA₄, and GA₈ compared with the Landsberg erecta wild type, whereas levels of immediate precursors to these GAs were elevated (Talon et al., 1990). Furthermore, metabolism of [¹³C]GA₂₀ to [¹³C]GA₁ was substantially less in the mutant than in the wild type (Kobayashi et al., 1994). In the present paper we confirm by functional expression of its cDNA in E. coli that GA4 encodes a GA 3β-hydroxylase. In addition, we determine the substrate specificity of recombinant GA4 using a number of C₂₀- and C₁₉-GAs and show by kinetic analysis that the enzyme has a higher affinity for GA₉ than for GA₂₀, which is consistent with the non-13-hydroxylation pathway predominating in Arabidopsis (Talon et al., 1990).     

Evolution of Primate Gene Expression: Drift and Corrective Sweeps?

Changes in gene expression play an important role in species' evolution. Earlier studies uncovered evidence that the effect of mutations on expression levels within the primate order is skewed, with many small downregulations balanced by fewer but larger upregulations. In addition, brain-expressed genes appeared to show an increased rate of evolution on the branch leading to human. However, the lack of a mathematical model adequately describing the evolution of gene expression precluded the rigorous establishment of these observations. Here, we develop mathematical tools that allow us to revisit these earlier observations in a model-testing and inference framework. We introduce a model for skewed gene-expression evolution within a phylogenetic tree and use a separate model to account for biological or experimental outliers. A Bayesian Markov chain Monte Carlo inference procedure allows us to infer the phylogeny and other evolutionary parameters, while quantifying the confidence in these inferences. Our results support previous observations; in particular, we find strong evidence for a sustained positive skew in the distribution of gene-expression changes in primate evolution. We propose a “corrective sweep” scenario to explain this phenomenon.     

Learning/Memory Impairment and Reduced Expression of the HNK-1 Carbohydrate in ��4-Galactosyltransferase-II-deficient Mice

The glycosylation of glycoproteins and glycolipids is important for central nervous system development and function. Although the roles of several carbohydrate epitopes in the central nervous system, including polysialic acid, the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides, have been investigated, those of the glycan backbone structures, such as Galβ1-4GlcNAc and Galβ1-3GlcNAc, are not fully examined. Here we report the generation of mice deficient in β4-galactosyltransferase-II (β4GalT-II). This galactosyltransferase transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the Gal β1-4GlcNAc structure, and it is strongly expressed in the central nervous system. In behavioral tests, the β4GalT-II^-/- mice showed normal spontaneous activity in a novel environment, but impaired spatial learning/memory and motor coordination/learning. Immunohistochemistry showed that the amount of HNK-1 carbohydrate was markedly decreased in the brain of β4GalT-II^-/- mice, whereas the expression of polysialic acid was not affected. Furthermore, mice deficient in glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of the HNK-1 carbohydrate, also showed impaired spatial learning/memory as described in our previous report, although their motor coordination/learning was normal as shown in this study. Histological examination showed abnormal alignment and reduced number of Purkinje cells in the cerebellum of β4GalT-II^-/- mice. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that the glycans synthesized by β4GalT-II have essential roles in higher brain functions, including some that are HNK-1-dependent and some that are not.The glycosylation of glycoproteins, proteoglycans, and glycolipids is important for their biological activities, stability, transport, and clearance from circulation, and cell-surface glycans participate in cell-cell and cell-extracellular matrix interactions. In the central nervous system, several specific carbohydrate epitopes, including polysialic acid (PSA),³ the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides play indispensable roles in neuronal generation, cell migration, axonal outgrowth, and synaptic plasticity (1). Functional analyses of the glycan backbone structures, like lactosamine core (Galβ1-4GlcNAc), neolactosamine core (Galβ1-3GlcNAc), and polylactosamine (Galβ1-4GlcNAcβ1-3) have been carried out using gene-deficient mice in β4-galactosyltransferase-I (β4GalT-I) (2, 3), β4GalT-V (4), β3-N-acetylglucosaminyl-transferase-II (β3GnT-II) (5), β3GnT-III (Core1-β3GnT) (6), β3GnT-V (7), and Core2GnT (8). However, the roles of these glycan backbone structures in the nervous system have not been examined except the olfactory sensory system (9).β4GalTs synthesize the Galβ1-4GlcNAc structure via the β4-galactosylation of glycoproteins and glycolipids; the β4GalTs transfer galactose (Gal) from UDP-Gal to a nonreducing terminal N-acetylglucosamine (GlcNAc) of N- and O-glycans with a β-1,4-linkage. The β4GalT family has seven members (β4GalT-I to VII), of which at least five have similar Galβ1-4GlcNAc-synthesizing activities (10, 11). Each β4GalT has a tissue-specific expression pattern and substrate specificity with overlapping, suggesting each β4GalT has its own biological role as well as redundant functions. β4GalT-I and β4GalT-II share the highest identity (52% at the amino acid level) among the β4GalTs (12), suggesting these two galactosyltransferases can compensate for each other. β4GalT-I is strongly and ubiquitously expressed in various non-neural tissues, whereas β4GalT-II is strongly expressed in neural tissues (13, 14). Indeed, the β4GalT activity in the brain of β4GalT-I-deficient (β4GalT-I^-/-) mice remains as high as 65% of that of wild-type mice, and the expression levels of PSA and the HNK-1 carbohydrate in the brain of these mice are normal (15). These results suggest β4GalTs other than β4GalT-I, like β4GalT-II, are important in the nervous system.Among the β4GalT family members, only β4GalT-I^-/- mice have been examined extensively; this was done by us and another group. We reported that glycans synthesized by β4GalT-I play various roles in epithelial cell growth and differentiation, inflammatory responses, skin wound healing, and IgA nephropathy development (2, 16-18). Another group reported that glycans synthesized by β4GalT-I are involved in anterior pituitary hormone function and in fertilization (3, 19). However, no other nervous system deficits have been reported in these mice, and the role of the β4-galactosylation of glycoproteins and glycolipids in the nervous system has not been fully examined.In this study, we generated β4GalT-II^-/- mice and examined them for behavioral abnormalities and biochemical and histological changes in the central nervous system. β4GalT-II^-/- mice were impaired in spatial learning/memory and motor coordination/learning. The amount of HNK-1 carbohydrate was markedly decreased in the β4GalT-II^-/- brain, but PSA expression was not affected. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that glycans synthesized by β4GalT-II have essential roles in higher brain functions, including ones that are HNK-1 carbohydrate-dependent and ones that are independent of HNK-1.     

Why Is the Correlation between Gene Importance and Gene Evolutionary Rate So Weak?

One of the few commonly believed principles of molecular evolution is that functionally more important genes (or DNA sequences) evolve more slowly than less important ones. This principle is widely used by molecular biologists in daily practice. However, recent genomic analysis of a diverse array of organisms found only weak, negative correlations between the evolutionary rate of a gene and its functional importance, typically measured under a single benign lab condition. A frequently suggested cause of the above finding is that gene importance determined in the lab differs from that in an organism's natural environment. Here, we test this hypothesis in yeast using gene importance values experimentally determined in 418 lab conditions or computationally predicted for 10,000 nutritional conditions. In no single condition or combination of conditions did we find a much stronger negative correlation, which is explainable by our subsequent finding that always-essential (enzyme) genes do not evolve significantly more slowly than sometimes-essential or always-nonessential ones. Furthermore, we verified that functional density, approximated by the fraction of amino acid sites within protein domains, is uncorrelated with gene importance. Thus, neither the lab-nature mismatch nor a potentially biased among-gene distribution of functional density explains the observed weakness of the correlation between gene importance and evolutionary rate. We conclude that the weakness is factual, rather than artifactual. In addition to being weakened by population genetic reasons, the correlation is likely to have been further weakened by the presence of multiple nontrivial rate determinants that are independent from gene importance. These findings notwithstanding, we show that the principle of slower evolution of more important genes does have some predictive power when genes with vastly different evolutionary rates are compared, explaining why the principle can be practically useful despite the weakness of the correlation.     

Differential Gene Expression in Thrombomodulin (TM; CD141)+ and TM? Dendritic Cell Subsets

Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin⁺ dendritic cells are tolerogenic while thrombomodulin⁻ dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin⁺ and thrombomodulin⁻ dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin⁺ dendritic cells compared to control dendritic cells and thrombomodulin⁻ dendritic cells. Thrombomodulin-treated and thrombomodulin⁺ dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin⁺ dendritic cells produced more lipoxins than thrombomodulin⁻ dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin⁺ dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin⁺ dendritic cells contained more cells actively dividing than those of thrombomodulin⁻ dendritic cells. Production of IL-10 is increased in thrombomodulin⁺ dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin⁺ dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin⁺ dendritic cells, which have significantly altered gene expression compared to thrombomodulin⁻ dendritic cells in key immune function pathways.     

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.