A model for diabetic nephropathy: advantages of the inducible cAMP early repressor transgenic mouse over the streptozotocin-induced diabetic mouse

We have previously found progressive diabetic nephropathy in inducible cAMP early repressor (ICER Igamma) transgenic (Tg) mice. The ICER Igamma Tg mouse is an interesting model of sustained hyperglycemia due to its low production of insulin and insulin-producing beta cells. Here in a longitudinal study we further analyzed diabetic nephropathy and structural and functional alterations in other organs, comparing our model with streptozotocin (STZ)-diabetic model mice. The high-dose STZ-diabetic model showed marked variation in blood glucose levels and severe toxicity of STZ in the liver and kidney. The low-dose STZ-diabetic model showed less toxicity, but the survival rate was very low. STZ-diabetic mice had much more variation of glomerular hypertrophy and sclerosis. Furthermore, non-specific toxicity of STZ or insulin injections to maintain optimal blood glucose levels might have another effect upon the diabetic renal changes. In contrast, ICER Igamma Tg mice exhibited a stable and progressive phenotype of diabetic kidney disease solely due to chronic hyperglycemia without other modulating factors. Thus, ICER Igamma Tg mouse has advantages for examining diabetic renal disease, and offers unique and very different perspectives compared to STZ model.     

Electrodialytic separation of levulinic acid catalytically synthesized from woody biomass for use in microbial conversion

Levulinic acid (LA) is produced by the catalytic conversion of a variety of woody biomass. To investigate the potential use of desalting electrodialysis (ED) for LA purification, electrodialytic separation of levulinate from both reagent and cedar‐derived LA solution (40–160 g L^?1) was demonstrated. When using reagent LA solution with pH5.0–6.0, the recovery rates of levulinate ranged from 68 to 99%, and the energy consumption for recovery of 1 kg of levulinate ranged from 0.18 to 0.27 kWh kg^?1. With cedar‐derived LA solution (pH6.0), good agreement in levulinate recovery (88–99%), and energy consumption (0.18–0.22 kWh kg^?1) were observed in comparison to the reagent LA solutions, although a longer operation time was required due to some impurities. The application of desalting ED was favorable for promoting microbial utilization of cedar‐derived LA. From 0.5 mol L^?1 of the ED‐concentrated sodium levulinate solution, 95.6% of levulinate was recovered as LA calcium salt dihydrate by crystallization. This is the first report on ED application for LA recovery using more than 20 g L^?1 LA solutions (40–160 g L^?1). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:448–453, 2017     

Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis

Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5′ leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5′ processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5′ leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.     

Linkage of Amino Acid Variation and Evolution of Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein (Subtype B) with Usage of the Second Receptor

To clarify the relationship between the amino acid variations of the gp120 of human immunodeficiency virus type 1 (HIV-1) and the chemokine receptors that are used as the second receptor for HIV, we evaluated amino acid site variation of gp120 between the X4 strains (use CXCR4) and the R5 strains (use CCR5) from 21 sequences of subtype B. Our analysis showed that residues 306 and 322 in the V3 loop and residue 440 in the C4 region were associated with usage of the second receptor. The polymorphism at residue 440 is clearly associated with the usage of the second receptor: The amino acid at position 440 was a basic amino acid in the R5 strains, and a nonbasic and smaller amino acid in the X4 strains, while the V3 loop of the X4 strains was more basic than that of the R5 strains. This suggests that residue 440 in the C4 region, which is close to the V3 loop in the three-dimensional structure, is critical in determining which second receptor is used. Analysis of codon frequency suggests that, in almost all cases, the difference at residue 440 between basic amino acids in the R5 strains and nonbasic amino acids in the X4 strains could be due to a single nucleotide change. These findings predict that the evolutionary changes in amino acid residue 440 may be correlated with evolutionary changes in the V3 loop. One possibility is that a change in electric charge at residue 440 compensates for a change in electric charge in the V3 loop. The amino acid polymorphism at position 440 can be useful to predict the cell tropism of a strain of HIV-1 subtype B.     

Arabidopsis CYP735A1 and CYP735A2 encode cytokinin hydroxylases that catalyze the biosynthesis of trans-Zeatin

Cytokinins (CKs), a group of phytohormones, are adenine derivatives that carry either an isoprene-derived or an aromatic side chain at the N(6) terminus. trans-Zeatin (tZ), an isoprenoid CK, is assumed to play a central physiological role because of its general occurrence and high activity in bioassays. Although hydroxylation of isopentenyladenine-type CKs is a key step of tZ biosynthesis, the catalyzing enzyme has not been characterized yet. Here we demonstrate that CYP735A1 and CYP735A2 are cytochrome P450 monooxygenases (P450s) that catalyze the biosynthesis of tZ. We identified the genes from Arabidopsis using an adenosine phosphate-isopentenyltransferase (AtIPT4)/P450 co-expression system in yeast. Co-expression of AtIPT4 and CYP735A enabled yeast to excrete tZ and the nucleosides to the culture medium. In vitro, both CYP735As preferentially utilized isopentenyladenine nucleotides rather than the nucleoside and free base forms and produced tZ nucleotides but not the cis-isomer. The expression of CYP735A1 and CYP735A2 was differentially regulated in terms of organ specificity and response to CK. Root-specific induction of CYP735A2 expression by CK suggests that the trans-hydroxylation is involved in the regulation of CK metabolism and signaling in roots.     

A rice antisense SPK transformant that lacks the accumulation of seed storage substances shows no correlation between sucrose concentration in phloem sap and demand for carbon sources in the sink organs

Rice SPK is a calmodulin-like domain protein kinase specific to immature seeds and promotes the degradation of sucrose. Therefore, antisense SPK transformants showed a defective production of storage starch, but accumulated sucrose in watery seeds. Despite a reduced sink strength, no difference was found in the sucrose concentration in phloem sap of the transformants and wild-type plants, which increased after floral organ induction to levels greater than 500 mM. However, sucrose was detected at relatively lower levels in the watery seed sap. These results suggest that sucrose content in the phloem is regulated independently from the demand for carbon sources in the sink organs.