Cloning and expression of a serine racemase gene homologue of the green alga Chlamydomonas reinhardtii and characterization of the gene product

A unicellular green alga Chlamydomonas reinhardtii (C. reinhardtii) has served as a model system to study many fundamental biological processes. We demonstrated that some D-amino acids have no inhibitory effect on the growth of C. reinhardtii and the green alga has alanine racemase and D-threonine aldolase. The homologous gene of serine racemase was found on the genome sequence of C. reinhardtii. In this study, a homologous gene of serine racemase on the genome of C. reinhardtii was cloned and expressed in E. coli cells, and the gene product was purified and characterized. Total RNA was extracted from C. reinhardtii cells. Sense and antisense primers were designed for PCR based on the upstream and downstream regions of the putative gene for serine racemase. First strand cDNA was synthesized from the mRNA and the antisense primer. Amplification of nucleotides between the two primers was performed with the cDNA. The fragment (ser-h) was sequenced. The deduced protein consisted of 340 amino acids with a molecular weight of 35,300. The amino acid sequence of the protein showed similarities to the reported serine racemases; Oryza sativa, 55%; Mus musculus, 52%; Schizosaccharomyces pombe, 39%. A modified serine racemase homologous (ser-h\u27) whose codons were optimized for E. coli was synthesized and used to construct pET24/ser-h\u27 and to transform BL21 (DE3). SDS-PAGE of the crude extract revealed that the gene product was overexpressed. The gene product was purified to electrophoretic homogeneity from the recombinant cells using ammonium sulfate fractionation and Column chromatography. Further characterization and crystallization of the enzyme are currently under study

Purification and pressure dependence of alanine racemase from the psychro- piezophilic bacterium shewanella violacea DSS 12

Shewanella violacea DSS12 (S. violacea) is a psychrophilic and piezophilic bacterium, isolated from mud of the Ryukyu Trench in Japan. The bacterium displays optimal growth at 8°C and 30 MPa. Alanine racemase is an enzyme which catalyses the interconversion of l-alanine and d-alanine, and is responsible for the synthesis of d-alanine contained in the peptidoglycan of bacterial cell wall. In this study, we purified alanine racemase from S. violacea and investigated the enzymological characteristics of alanine racemase. The bacterium was aerobically cultured using marine broth 2216 in a 5-liter medium bottle at 4°C for 3 days. The bacterial cells were lysed by applying of 100 MPa pressure using a French press, and the lysate was centrifuged. The supernatant obtained was ultracentrifuged at 141,000 g, and the supernatant obtained was applied to ammonium sulfate fractionation. The active fraction was dissolved and passed through a butyl-Toyopearl, phenyl-Sepharose, and shodex KW-200 columns to obtain a partially purified enzyme. Consequently, the enzyme was purified 540-fold and showed a specific activity of 2.68 μmol/min/mg. Alanine racemase exhibited high activity against l-Ala and l-Ser as substrates. The optimal pH and temperature of alanine racemase were 9.0 and 25°C, respectively. Please click Additional Files below to see the full abstract

1-year tolvaptan efficacy in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) develops into end-stage kidney disease by 65 years of age in an estimated 45%-70% of patients. Recent trials revealed that tolvaptan inhibits disease progression both in early-stage or late-stage ADPKD ; however, stratified analysis showed a difference of favorable factors correlated with tolvaptan efficacy between early-stage and late-stage ADPKD. Thus, we examined the efficacy of tolvaptan in ADPKD with a wide range of estimated glomerular filtration rates (eGFR). We enrolled 24 patients with eGFR 35.3 (28.0-65.5) ml / min / 1.73m2 and evaluated treatment effect as ΔΔeGFR (ml / min / 1.73m2 / year) or ΔΔtotal kidney volume (TKV) (% / year) that was calculated as post-treatment annual change - pre-treatment annual change. Pre ΔeGFR was significantly low in eGFR responders, defined as ΔΔeGFR > 0 ml / min / 1.73m2 / year. In eGFR responders, pre ΔeGFR, post ΔeGFR, eGFR, TKV, and proteinuria were significantly correlated with ΔΔeGFR. In TKV responders defined as ΔΔTKV > 5 % / year, we identified hypertension history, proteinuria, TKV, and post ΔTKV as significantly correlated factors with ΔΔTKV. In conclusion, pre ΔeGFR may be a predictive factor of therapeutic efficacy on kidney function. Tolvaptan may have greater efficacy in early-stage ADPKD with rapid GFR decline or with well-controlled blood pressure

Callose-mediated resistance to pathogenic intruders in plant defense-related papillae

Plants are exposed to a wide range of potential pathogens, which derive from diverse phyla. Therefore, plants have developed successful defense mechanisms during co-evolution with different pathogens. Besides many specialized defense mechanisms, the plant cell wall represents a first line of defense. It is actively reinforced through the deposition of cell wall appositions, so-called papillae, at sites of interaction with intruding microbial pathogens. The papilla is a complex structure that is formed between the plasma membrane and the inside of the plant cell wall. Even though the specific biochemical composition of papillae can vary between different plant species, some classes of compounds are commonly found which include phenolics, reactive oxygen species, cell wall proteins, and cell wall polymers. Among these polymers, the (1,3)-β-glucan callose is one of the most abundant and ubiquitous components. Whereas the function of most compounds could be directly linked with cell wall reinforcement or an anti-microbial effect, the role of callose has remained unclear. An evaluation of recent studies revealed that the timing of the different papilla-forming transport processes is a key factor for successful plant defense

Network Properties of Robust Immunity in Plants

Two modes of plant immunity against biotrophic pathogens, Effector Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), are triggered by recognition of pathogen effectors and Microbe-Associated Molecular Patterns (MAMPs), respectively. Although the jasmonic acid (JA)/ethylene (ET) and salicylic acid (SA) signaling sectors are generally antagonistic and important for immunity against necrotrophic and biotrophic pathogens, respectively, their precise roles and interactions in ETI and PTI have not been clear. We constructed an Arabidopsis dde2/ein2/pad4/sid2-quadruple mutant. DDE2, EIN2, and SID2 are essential components of the JA, ET, and SA sectors, respectively. The pad4 mutation affects the SA sector and a poorly characterized sector. Although the ETI triggered by the bacterial effector AvrRpt2 (AvrRpt2-ETI) and the PTI triggered by the bacterial MAMP flg22 (flg22-PTI) were largely intact in plants with mutations in any one of these genes, they were mostly abolished in the quadruple mutant. For the purposes of this study, AvrRpt2-ETI and flg22-PTI were measured as relative growth of Pseudomonas syringae bacteria within leaves. Immunity to the necrotrophic fungal pathogen Alternaria brassicicola was also severely compromised in the quadruple mutant. Quantitative measurements of the immunity levels in all combinatorial mutants and wild type allowed us to estimate the effects of the wild-type genes and their interactions on the immunity by fitting a mixed general linear model. This signaling allocation analysis showed that, contrary to current ideas, each of the JA, ET, and SA signaling sectors can positively contribute to immunity against both biotrophic and necrotrophic pathogens. The analysis also revealed that while flg22-PTI and AvrRpt2-ETI use a highly overlapping signaling network, the way they use the common network is very different: synergistic relationships among the signaling sectors are evident in PTI, which may amplify the signal; compensatory relationships among the sectors dominate in ETI, explaining the robustness of ETI against genetic and pathogenic perturbations

Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca2+. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca2+ link between MAMP recognition and SA accumulation that is important for resistance to P. syringae