An Improved Fluorometric High-Performance Liquid Chromatography Method for Sialic Acid Determination: An Internal Standard Method and Its Application to Sialic Acid Analysis of Human Apolipoprotein E

An improved fluorometric HPLC method for sialic acid determination was developed by employing synthetic N-propionylneuraminic acid (NPNA) as an internal standard. A fixed amount of NPNA was added to a sialoglycoconjugate sample. After hydrolyzing sialioglycoconjugates with diluted sulfuric acid, the released sialic acids and NPNA were derivatized with a fluorogenic compound, 1,2-diamino-4,5-(methylenedioxy)benzene (DMB), followed by fluorometric HPLC. The fluorescent derivative of NPNA was separated from those of N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid, and 2-keto-3-deoxyoctanoate on HPLC. The separation of NPNA derivative on HPLC was not interfered by components of biological samples such as human sera. Using this internal standard method, low amounts of NANA (0.15-1.0 ng) were quantified with the coefficient of variation values below 4%. Using this method, the sialic acid content of human apolipoprotein E was successfully determined. The present method is useful for sensitive and accurate quantification of sialic acids of different molecular species in biological samples.     

A retrospective analysis of germline competence in rat embryonic stem cell lines

The factors responsible for conferring germline competence in embryonic stem (ES) cell lines remain unidentified. In the present study, rat ES cell lines (n = 17) were established with 3i medium (SU5402, PD0325901, CHIR99021), 2i medium (PD0325901, CHIR99021) or 2iF medium (PD0325901, CHIR99021, forskolin), and their potential for germline transmission to the G1 generation was examined. Rat strains were divided into an albino group (F344, Wistar or CAG/Venus transgenic rats with the Wistar background) or a colored coat group (Brown-Norway, Dark-Agouti, or BLK rats selected from >F3 generations of Wistar × Dark-Agouti rats based on their black coat color). Successful germline transmission was observed in 57 % (4/7), 40 % (2/5) and 100 % (5/5) of the ES cells established with 3i, 2i and 2iF media, respectively. ES cell lines from the homozygous CAG/Venus transgenic rats were established in all three media, but only the lines established with the 2iF medium were germline-competent. Neither coat-color (albino: 64 %, 7/11; colored: 67 %, 4/6) nor gender of the ES cell lines (XX: 67 %, 2/3; XY: 64 %, 9/14) were likely to affect germline transmission.     

Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum

Protein Nε‐acylation is emerging as a ubiquitous post‐translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of l ‐glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction was characterized. It was shown that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation‐mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine‐incorporated PEPC protein, we verified that K653‐acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin‐type deacetylase, deacetylated K653‐acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin‐type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate‐producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate‐producing conditions, supporting the hypothesis that PEPC is responsible for a large carbon flux change under glutamate‐producing conditions.     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

Effects of mutation of Asn694 in Aspergillus niger α-glucosidase on hydrolysis and transglucosylation

Aspergillus niger α-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of α-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of α-(1→6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(α1–4)Glc] yields both α-(1→6)- and α-(1→4)-glucosidic linkages, the latter constituting ~25% of the total transfer reaction product. The maltotriose [Glc(α1–4)Glc(α1–4)Glc], α-(1→4)-glucosyl product disappears quickly, whereas the α-(1→6)-glucosyl products panose [Glc(α1–6)Glc(α1–4)Glc], isomaltose [Glc(α1–6)Glc], and isomaltotriose [Glc(α1–6)Glc(α1–6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the α-(1→4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).

     

Purification and characterization of macrolide 2''-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Hyperimmune and high-titered polyclonal pneumococcal antisera, specific for cross-reactive types within groups, were produced in adult rabbits. Purified capsular polysaccharide was injected intravenously into adult rabbits. One week later, these rabbits were given multiple intravenous injections of formalin-inactivated pneumococci of the cross-reactive type by an established method. Each of the resultant antisera were specific for the cross-reactive type indicating that the previous injection of the polysaccharide had induced epitope-specific tolerance. This method was successful for production of antisera against pneumococcal types 6A, 6B, 9N, 9V, 19F and 19A. Polyclonal rabbit pneumococcal antisera have some advantages over murine monoclonal antibodies for serologic studies and this method should be applicable for producing type-specific antibodies to cross-reactive polysaccharides of clinical interest. Further, this method is simpler and generally produces higher titered monovalent (factor) reagents than absorbed antisera.     

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

Relevance of Distinct Monocyte Subsets to Clinical Course of Ischemic Stroke Patients

Background and Purpose

The most common strategy for treating patients with acute ischemic stroke is thrombolytic therapy, though only a few patients receive benefits because of the narrow time window. Inflammation occurring in the central nervous system (CNS) in association with ischemia is caused by immune cells including monocytes and involved in lesion expansion. If the specific roles of monocyte subsets in stroke can be revealed, they may become an effective target for new treatment strategies.

Methods

We performed immunological examinations of 36 consecutive ischemic stroke patients within 2 days of onset and compared the results with 24 age-matched patients with degenerative disorders. The stroke patients were repeatedly tested for the proportions of monocyte subsets in blood, and serum levels of pro- and anti-inflammatory cytokines immediately after admission, on days 3-7 and 12-16 after stroke onset, and on the day of discharge. In addition, immunological measurements were analyzed for relationships to stroke subtypes and complications, including progressive infarction (PI) and stroke-associated infection (SAI).

Results

Monocyte count was significantly increased from 0–16 days after stroke as compared to the controls (p<0.05). CD14^highCD16^- classical and CD14^highCD16⁺ intermediate monocytes were significantly increased from 0-7 and 3-16 days after stroke, respectively (p<0.05), whereas CD14 ^dimCD16^high non-classical monocytes were decreased from 0–7 days (p<0.05). Cardioembolic infarction was associated with a persistent increase in intermediate monocytes. Furthermore, intermediate monocytes were significantly increased in patients with PI (p<0.05), while non-classical monocytes were decreased in those with SAI (p<0.05). IL-17A levels were positively correlated with monocyte count (r=0.485, p=0.012) as well as the percentage of non-classical monocytes (r=0.423, p=0.028), and negatively with that of classical monocytes (r=-0.51, p=0.007) during days 12-16.

Conclusions

Our findings suggest that CD14^highCD16⁺ intermediate monocytes have a role in CNS tissue damage during acute and subacute phases in ischemic stroke especially in relation to cardioembolism.