Stable expression of the sweet protein monellin variant MNEI in tobacco chloroplasts

Monellin is a naturally sweet protein that consists of two polypeptide chains and has potential uses as a highly potent non-carbohydrate sweetener. We aimed to make this protein more usable by increasing its stability and expressing it in a high-yielding system. MNEI is a modified version of the protein that consists of the two natural chains of monellin joined via a dipeptide linkage. In the thermostability analysis of MNEI variants, four mutated MNEIs, MNEI-E24L, MNEI-E24F, MNEI-E24W, and MNEI-E24A, had higher melting temperatures than wild-type MNEI and retained their sweet flavor even at temperatures above 70?°C. Our findings indicate that the increased stability of monellin allows it to retain its strong sweetness even under extreme conditions. We successfully overexpressed the thermostable MNEI mutants in tobacco chloroplasts. Here, we report that the MNEI mutants showed enhanced thermostability, and the stable forms of MNEI can be produced through plastid transformation in tobacco.     

Ceramide is a competitive inhibitor of diacylglycerol kinase in vitro and in intact human leukemia (HL-60) cells.

Prior studies demonstrated that ceramide was phosphorylated by a novel Ca(2+)-dependent kinase distinct from diacylglycerol (DG) kinase in human myelogenous leukemia (HL-60) cells (Kolesnick, R. N., and Hemer, M. R. (1990) J. Biol. Chem. 265, 10900-10904). The present studies were initiated to determine whether mammalian DG kinase purified to homogeneity possessed phosphotransferase activity toward ceramide. A high molecular weight rat brain DG kinase demonstrated Mg(2+)-(but not Ca(2+)-) dependent DG kinase activity and did not phosphorylate ceramide in the presence of either cation. In contrast, ceramide served as a competitive inhibitor with an inhibition constant (Ki) 2-6-fold greater than the Km for DG. Inhibition was noncompetitive with respect to ATP and Mg2+. A cell-permeable ceramide, N-octanoyl sphingosine (C8-cer), was used to study effects of ceramide on DG kinase in intact HL-60 cells. C8-cer induced dose- and time-dependent increases in cellular DG levels. As little as 1 microM C8-cer increased DG from a basal level of 103 to 177 pmol.10(6) cells-1, and a maximal 2.9-fold elevation to 292 pmol.10(6) cells-1 occurred with 10 microM C8-cer. DG elevation was detected after 1 min, maximal by 7.5 min, and sustained for 30 min. The DG elevation was accompanied by a reduction in 32P incorporation in phosphatidic acid in cells short term-labeled with [32P]orthophosphoric acid, consistent with inhibition of DG kinase. In contrast, a similar elevation in the DG level induced by exogenous phospholipase C increased 32P incorporation into phosphatidic acid. C8-cer was not metabolized to sphingomyelin, indicating that DG was not generated through the phosphatidylcholine:ceramide cholinephosphotransferase reaction. DG elevation after C8-cer or phospholipase C treatment was sufficient to redistribute protein kinase C from cytosol to membrane. These findings provide evidence that ceramide may serve as a competitive inhibitor of DG kinase.     

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacI^q, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.     

6′-Mercaptoguanosine-resistance is related with purF gene encoding 5′-phosphoribosyl-1′-pyrophosphate amidotransferase in inosine-5′-monophosphate overproducing Brevibacterium ammoniagenes

From the gene library constructed with the chromosomal DNA of 6-mercaptoguanosine (MGS)-resistant strain Brevibacterium ammoniagenes IPR-1, a DNA fragment which conferred MGS-resistance to the wild-type strain B. ammoniagenes ATCC6872 was cloned. The purF gene encoding 5-phosphoribosyl-1-pyrophosphate amidotransferase was identified from this fragment and its nucleotide sequence was determined. Wild type purF gene was also cloned by polymerase chain reaction using chromosomal DNA of ATCC6872 as the template and its sequence was determined. Two nucleotides, 583 A and 1065 A, of MGS-resistant purF gene had been changed from 583 G and 1065 G by mutagenesis, respectively. Both changes at position 583 and 1065 were proved to be responsible for MGS-resistance by site-directed mutagenesis.     

Glutaraldehyde crosslinking of red blood cells by using a hemodialyzer

Summary Human red blood cells (RBC) were crosslinked with glutaraldehyde (GA) by using a hemodialyzer which is used as an artificial kidney. Human RBC, which was in a flow of 2 ml/min, was extensively crosslinked with 50 mM GA solution of 10 ml/min flow rate. The crosslinked RBC showed high stability against osmotic pressure. The oxygen transport activity of the crosslinked RBC was similar to unmodified RBC. This crosslinking method could be used for the development of an efficient reactor which produces a stable and active RBC.     

Effect of Immunotherapy on Seizure Outcome in Patients with Autoimmune Encephalitis: A Prospective Observational Registry Study

Objective

To evaluate the seizure characteristics and outcome after immunotherapy in adult patients with autoimmune encephalitis (AE) and new-onset seizure.

Methods

Adult (age ≥18 years) patients with AE and new-onset seizure who underwent immunotherapy and were followed-up for at least 6 months were included. Seizure frequency was evaluated at 2–4 weeks and 6 months after the onset of the initial immunotherapy and was categorized as “seizure remission”, “> 50% seizure reduction”, or “no change” based on the degree of its decrease.

Results

Forty-one AE patients who presented with new-onset seizure were analysed. At 2–4 weeks after the initial immunotherapy, 51.2% of the patients were seizure free, and 24.4% had significant seizure reduction. At 6 months, seizure remission was observed in 73.2% of the patients, although four patients died during hospitalization. Rituximab was used as a second-line immunotherapy in 12 patients who continued to have seizures despite the initial immunotherapy, and additional seizure remission was achieved in 66.6% of them. In particular, those who exhibited partial response to the initial immunotherapy had a better seizure outcome after rituximab, with low adverse events.

Conclusion

AE frequently presented as seizure, but only 18.9% of the living patients suffered from seizure at 6 months after immunotherapy. Aggressive immunotherapy can improve seizure outcome in patients with AE.     

Prognostic Value of SUVmax Measured by Pretreatment Fluorine-18 Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography in Patients with Ewing Sarcoma

AimThe aim of this retrospective study was to determine whether glucose metabolism assessed by using Fluorine-18 (F-18) fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) provides prognostic information independent of established prognostic factors in patients with Ewing sarcoma.MethodsWe retrospectively reviewed the medical records of 34 patients (men, 19; women, 15; mean age, 14.5 ± 9.7 years) with pathologically proven Ewing sarcoma. They had undergone F-18 FDG PET/CT as part of a pretreatment workup between September 2006 and April 2012. In this analysis, patients were classified by age, sex, initial location, size, and maximum standardized uptake value (SUVmax). The relationship between FDG uptake and survival was analyzed using the Kaplan-Meier method with the log-rank test and Cox’s proportional hazards regression model.ResultsThe median survival time for all 34 subjects was 999 days and the median SUV by using PET/CT was 5.8 (range, 2–18.1). Patients with a SUVmax ≤ 5.8 survived significantly longer than those with a SUVmax > 5.8 (median survival time, 1265 vs. 656 days; p = 0.002). Survival was also found to be significantly related to age (p = 0.024), size (p = 0.03), and initial tumor location (p = 0.036). Multivariate analysis revealed that a higher SUVmax (p = 0.003; confidence interval [CI], 3.63–508.26; hazard ratio [HR], 42.98), older age (p = 0.023; CI, 1.34–54.80; HR, 8.59), and higher stage (p = 0.03; CI, 1.21–43.95; HR, 7.3) were associated with worse overall survival.ConclusionsSUVmax measured by pretreatment F-18-FDG PET/CT can predict overall survival in patients with Ewing sarcoma.     

Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis.

The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues. The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN' and 70% with subtilisin DY. Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. It retains about 50% of its activity even after treatment at 60 degrees C for 30 min in the presence of 2 mM calcium chloride.     

Sulfhydryl groups are essential for organic cation exchange in rabbit renal basolateral membrane vesicles

The effect of N-ethylmaleimide (NEM), an irreversible sulfhydryl modifying reagent, on the transport of organic cations in the renal basolateral membrane was examined. The studies were conducted examining the exchange of [3H]tetraethylammonium (TEA) for unlabeled TEA in basolateral membrane vesicles isolated from the outer cortex of rabbit kidneys. NEM inactivated TEA transport in a dose-dependent fashion with an IC50 value of 260 microM. The rate of TEA transport inactivation followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 4.0. The data imply that inactivation involves the binding of at least four molecules of NEM per active transport unit. This is most consistent with the presence of four sulfhydryl groups at this site. The substrate TEA displayed a dose-dependent enhancement of NEM inactivation, with 50% enhancement occurring at 365 microM TEA. Another organic cation, N1-methylnicotinamide, known to share a common transport mechanism with the TEA/TEA exchanger is also capable of increasing the reactivity of sulfhydryl groups to NEM. These results demonstrate that there are essential sulfhydryl groups for organic cation transport in the basolateral membrane. In addition, the capability of organic cations to alter the susceptibility to sulfhydryl modification suggests that these groups may have a dynamic role in the transport process.