Glucokinase Variant Proteins Are Resistant to Fasting-Induced Uridine Diphosphate Glucose-Dependent Degradation in Maturity-Onset Diabetes of the Young Type 2 Patients.

We previously reported that glucokinase undergoes ubiquitination and subsequent degradation, a process mediated by cereblon, particularly in the presence of uridine diphosphate glucose (UDP-glucose). In this context, we hereby present evidence showcasing the resilience of variant glucokinase proteins of maturity-onset diabetes of the young type 2 (MODY2) against degradation and, concomitantly, their influence on insulin secretion, both in cell lines and in the afflicted MODY2 patient. Hence, glucose-1-phodphate promotes UDP-glucose production by UDP-glucose pyrophosphorylase 2; consequently, UDP-glucose-dependent glucokinase degradation may occur during fasting. Next, we analyzed glucokinase variant proteins from MODY2 or persistent hyperinsulinemic hypoglycemia in infancy (PHHI). Among the eleven MODY2 glucokinase-mutated proteins tested, those with a lower glucose-binding affinity exhibited resistance to UDP-glucose-dependent degradation. Conversely, the glucokinaseA456V-mutated protein from PHHI had a higher glucose affinity and was sensitive to UDP-glucose-dependent degradation. Furthermore, in vitro studies involving UDP-glucose-dependent glucokinase variant proteins and insulin secretion during fasting in Japanese MODY2 patients revealed a strong correlation and a higher coefficient of determination. This suggests that UDP-glucose-dependent glucokinase degradation plays a significant role in the pathogenesis of glucose-homeostasis-related hereditary diseases, such as MODY2 and PHHI.

R1526 residue in arginine/proinsulin binding domain of UGGT1 is involved in proinsulin binding.

Arginine releases proinsulin from binding to UGGT1 in the endoplasmic reticulum (ER). PSIPRED analysis showed that the arginine/proinsulin binding domain (A/PBD) in the C-terminal region of UGGT1 forms a disordered region, which is flexible and outside of the main protein structure. Both arginine and proinsulin may easily access the disordered region of A/PBD. Using the SNP library, two variants, Q1518∗ and R1526C, were identified in this region. UGGT1Q1518∗ protein is a deficient form of A/PBD and ER-retention signal (ERRS). UGGT1Q1518∗ protein in cell analysis reveals that mutated protein is mainly secreted from the cells because it lacks ERRS. We found another UGGT1 variant, UGGT1R1526C. At the molecular level, less interaction of proinsulin with UGGT1 was observed in both human UGGT1R1526C and mouse UGGT1L1518C with/without arginine. However, UGGT1R1526C and UGGT1WT interact with arginine similarly. We identified several amino acid residues for the arginine and proinsulin interaction. Here, the R1526 residue of UGGT1 is involved in proinsulin-interaction and is not involved in arginine-interaction.

Efficacy of a newly developed bioabsorbable pancreatic clip for distal pancreatectomy in swine.

PURPOSE: At present, ≥ 20% of patients experience clinically relevant postoperative pancreatic fistula (POPF) after distal pancreatectomy (DP). METHODS: We developed a new bioabsorbable pancreatic clip (BioPaC) made of polycaprolactone that does not crush the pancreatic parenchyma during occlusion of the pancreatic stump. We confirmed the efficacy of this BioPac in a porcine DP model and compared it to a linear stapling device (Reinforce®). RESULTS: Pigs were killed at 1 month after DP. In the BioPaC group, all swine (n = 3) survived well without POPF. In the Reinforce® group (n = 2), one pig died early at postoperative day 7 with Grade C POPF (amylase 43 700 U/l), and the other survived until 1 month at scarification with biochemical leakage of POPF (amylase 3 725 U/l). Pathologically, the main pancreatic duct and pancreatic parenchyma were well closed by BioPaC. CONCLUSION: The newly developed BioPaC is effective in a porcine DP model.

UDP-Glucose: A Cereblon-Dependent Glucokinase Protein Degrader.

We previously reported that glucokinase is ubiquitinated and degraded by cereblon with an unknown endogenous glucokinase protein degrader. Here, we show that UDP-glucose is a glucokinase protein degrader. We identified that both glucose and UDP-glucose bind to glucokinase and that both uridine and UDP-glucose bind to cereblon in a similar way to thalidomide. From these results, UDP-glucose was identified as a molecular glue between cereblon and glucokinase. Glucokinase produces glucose-6-phosphate in the pancreas and liver. Especially in ß-cells, glucokinase is the main target of glucose for glucose-induced insulin secretion. UDP-glucose administration ubiquitinated and degraded glucokinase, lowered glucose-6-phosphate production, and then reduced insulin secretion in ß-cell lines and mice. Maturity-onset diabetes of the young type 2 (MODY2) glucokinaseE256K mutant protein was resistant to UDP-glucose induced ubiquitination and degradation. Taken together, glucokinase ubiquitination and degradation signaling might be impaired in MODY2 patients.

SlHair2 Regulates the Initiation and Elongation of Type I Trichomes on Tomato Leaves and Stems.

Trichomes are hair-like structures that are essential for abiotic and biotic stress responses. Tomato Hair (H), encoding a C2H2 zinc finger protein, was found to regulate the multicellular trichomes on stems. Here, we characterized Solyc10g078990 (hereafter Hair2, H2), its closest homolog, to examine whether it was involved in trichome development. The H2 gene was highly expressed in the leaves, and its protein contained a single C2H2 domain and was localized to the nucleus. The number and length of type I trichomes on the leaves and stems of knock-out h2 plants were reduced when compared to the wild-type, while overexpression increased their number and length. An auto-activation test with various truncated forms of H2 using yeast two-hybrid (Y2H) suggested that H2 acts as a transcriptional regulator or co-activator and that its N-terminal region is important for auto-activation. Y2H and pull-down analyses showed that H2 interacts with Woolly (Wo), which regulates the development of type I trichomes in tomato. Luciferase complementation imaging assays confirmed that they had direct interactions, implying that H2 and Wo function together to regulate the development of trichomes. These results suggest that H2 has a role in the initiation and elongation of type I trichomes in tomato.

Chorionic gonadotropin stimulates maternal hepatocyte proliferation during pregnancy.

The liver increases its size during pregnancy to adapt to metabolic demand associated with pregnancy. Our previous study showed that proliferation of maternal hepatocytes are increased during pregnancy in mice and that estradiol (E2) is one of the candidate hormones responsible for maternal hepatocyte proliferation. Here, we discovered that chorionic gonadotropin (CG) induces maternal hepatocyte proliferation during pregnancy. CG administration was sufficient to stimulate hepatocyte proliferation in non-pregnant mice as well as in cell culture system. We conclude that CG stimulates proliferation in the early pregnancy of maternal hepatocytes. In contrast, estrogen stimulates hepatocyte proliferation in the late pregnancy.

Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana.

Growth plasticity is a key mechanism by which plants adapt to the ever-changing environmental conditions. Since growth is a high-energy-demanding and irreversible process, it is expected to be regulated by the integration of endogenous energy status as well as environmental conditions. Here, we show that trehalose-6-phosphate (T6P) functions as a sugar signaling molecule that coordinates thermoresponsive hypocotyl growth with endogenous sugar availability. We found that the loss of T6P SYNTHASE 1 (TPS1) in Arabidopsis thaliana impaired high-temperature-mediated hypocotyl growth. Consistently, the activity of PIF4, a transcription factor that positively regulates hypocotyl growth, was compromised in the tps1 mutant. We further show that, in the tps1 mutant, a sugar signaling kinase KIN10 directly phosphorylates and destabilizes PIF4. T6P inhibits KIN10 activity in a GRIK-dependent manner, allowing PIF4 to promote hypocotyl growth at high temperatures. Together, our results demonstrate that T6P determines thermoresponsive growth through the KIN10-PIF4 signaling module. Such regulation of PIF4 by T6P integrates the temperature-signaling pathway with the endogenous sugar status, thus optimizing plant growth response to environmental stresses.

Does an "Off-Track" Hill-Sachs Lesion That Is Misclassified as "Non-Engaging" Affect Outcomes From Bankart Repair Alone Compared With Bankart Repair Combined With Remplissage?

PURPOSE: To determine how intraoperative assessment (engagement test) may affect recurrent dislocation rate and to compare the clinical outcomes, recurrence rates, and presence of on-/off-track conditions between cases that received arthroscopic Bankart repair alone (nonengaged Hill-Sachs lesion) and Bankart repair with remplissage (engaged Hill-Sachs lesion). METHODS: We retrospectively reviewed 213 patients who underwent arthroscopic Bankart repair alone (186 patients with nonengaging lesions, group A) or with remplissage (27 patients with engaging lesion, group B) for recurrent anterior shoulder instability with <25% glenoid bone defect. The presence of an engaging Hill-Sachs lesion was determined during arthroscopic evaluation. On-track or off-track lesions were assessed retrospectively from preoperative 3-dimensional (3D) computed tomography (CT). RESULTS: Mean glenoid bone defect was 13.7% in group A and 20.7% in group B (P < .001). Off-track lesions were identified in 8.1% (15/186) and 100% (27/27) in group B. At the final follow-up (minimum 2 years; mean follow-up periods after surgery of 50.1 months in group A and 47.7 months in group B), there were no significant differences in shoulder functional scores and recurrence rates between groups, despite improvement after surgery. In the off-track lesion (group A-1: nonengaging but off-track lesion), recurrence instability occurred in 9 patients (60%, 9/15). Also, comparing group A-1 and group B, we noted significant differences in shoulder functional scores and recurrence rates (P < .001). CONCLUSION: Of 186 patients, 8.1% with nonengaging Hill-Sachs lesions during direct arthroscopic examination under anesthesia actually demonstrated off-track lesions on preoperative 3D CT scans retrospectively, with 60% experiencing recurrent instability. Intraoperative manual assessment for Hill-Sachs engagement was inferior to 3D CT scan in establishing the presence of off-track defects. LEVEL OF EVIDENCE: III, retrospective comparative study.

UGGT1 retains proinsulin in the endoplasmic reticulum in an arginine dependent manner.

We sought to clarify a pathway by which L- and dD-arginine simulate insulin secretion in mice and cell lines and obtained the following novel two findings. (1) Using affinity magnetic nanobeads technology, we identified that proinsulin is retained in the endoplasmic reticulum (ER) through UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) when arginine availability is limited. (2) L- and d-arginine release proinsulin from UGGT1 through competition with proinsulin and promote exit of proinsulin from the ER to Golgi apparatus. The ability of arginine to release proinsulin from UGGT1 closely correlates with arginine-induced insulin secretion in several models of ß cells indicating that UGGT1-proinsulin interaction regulates arginine-induced insulin secretion.

A Randomized Phase II Study of FOLFOX With or Without the MET Inhibitor Onartuzumab in Advanced Adenocarcinoma of the Stomach and Gastroesophageal Junction.

BACKGROUND: The phase II YO28252 study (NCT01590719) examined first-line onartuzumab plus mFOLFOX6 in patients with metastatic, human epidermal growth factor receptor 2-negative adenocarcinoma of the stomach or gastroesophageal junction. MET immunohistochemistry expression as a biomarker of onartuzumab activity was also examined. PATIENTS AND METHODS: Patients were randomized 1:1 to receive standard mFOLFOX6 plus onartuzumab (10 mg/kg) or placebo in 2-week cycles for 12 cycles, followed by onartuzumab or placebo until disease progression. Coprimary endpoints were progression-free survival (PFS) in intent-to-treat (ITT) and MET-positive populations. The target hazard ratio (HR) was 0.70 for patients in the ITT group and 0.60 in the MET-positive population. Secondary endpoints were overall survival (OS), overall response rate (ORR), and safety. RESULTS: Overall, 123 patients were enrolled (n = 62 onartuzumab, n = 61 placebo). Median PFS was 6.77 versus 6.97 months for onartuzumab versus placebo, respectively (HR, 1.08; 95% confidence interval [CI], 0.71-1.63; p = .71). In the MET-positive population, median PFS was 5.95 versus 6.80 months, onartuzumab versus placebo (HR, 1.38; 95% CI, 0.60-3.20; p = .45). Median OS was 10.61 months for onartuzumab versus 11.27 months for placebo) (HR, 1.06, 0.64-1.75; p = .83). In the MET-positive population, median OS was 8.51 versus 8.48 months for onartuzumab versus placebo, respectively (HR, 1.12, 95% CI, 0.45-2.78; p = .80). ORR was 60.5% for the onartuzumab group and 57.1% for placebo. Grade 3-5 adverse events (AEs) were seen in 88.3% of patients receiving onartuzumab and in 78.3% of patients receiving placebo, with serious AEs in 55% and 40%, respectively. CONCLUSION: The addition of onartuzumab to mFOLFOX6 in gastric cancer did not improve efficacy in an unselected population or in a MET immunohistochemistry-positive population. IMPLICATIONS FOR PRACTICE: The YO28252 study demonstrated that the addition of the anti-MET agent onartuzumab to mFOLFOX6 for treatment of gastric cancer did not improve efficacy in an overall study population or those selected for positive MET status by immunohistochemistry. This highlights the importance of correctly selecting biomarkers for targeted therapies. A multivariate analysis suggested that MET positivity may still be prognostic for worse median overall survival in gastric cancer; therefore, it is important to continue investigation into the optimal approach to inhibit MET signaling in gastric cancer.

Biodegradable Inorganic Nanovector: Passive versus Active Tumor Targeting in siRNA Transportation.

The biodegradable inorganic nanovector based on a layered double hydroxide (LDH) holds great promise for gene and drug delivery systems. However, in vivo targeted delivery of genes through LDH still remains a key challenge in the development of RNA interference therapeutics. Here, we describe in vivo and in vitro delivery system for Survivin siRNA (siSurvivin) assembled with passive LDH with a particle size of 100 nm or active LDH conjugated with a cancer overexpressing receptor targeting ligand, folic acid (LDHFA), conferring them an ability to target the tumor by either EPR-based clathrin-mediated or folate receptor-mediated endocytosis. When not only transfected into KB cells but also injected into xenograft mice, LDHFA/siSurvivin induced potent gene silencing at mRNA and protein levels in vitro, and consequently achieved a 3.0-fold higher suppression of tumor volume than LDH/siSurvivin in vivo. This anti-tumor effect was attributed to a selectively 1.2-fold higher accumulation of siSurvivin in tumor tissue compared with other organs. Targeting to the tumor with inorganic nanovector can guide and accelerate an evolution of next-generation theranosis system.

The antifungal activity and membrane-disruptive action of dioscin extracted from Dioscorea nipponica.

Dioscin is a kind of steroidal saponin isolated from the root bark of wild yam Dioscorea nipponica. We investigated the antifungal effect of dioscin against different fungal strains and its antifungal mechanism(s) in Candida albicans cells. Using the propidium iodide assay and calcein-leakage measurement, we confirmed that dioscin caused fungal membrane damage. Furthermore, we evaluated the ability of dioscin to disrupt the plasma membrane potential, using 3,3'-dipropylthiadicarbocyanine iodide [DiSC(3)(5)] and bis-(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)]. Cells stained with the dyes had a significant increase in fluorescent intensity after exposure to dioscin, indicating that dioscin has an effect on the membrane potential. To visualize the effect of dioscin on the cell membrane, we synthesized rhodamine-labeled giant unilamellar vesicles (GUVs) mimicking the outer leaflet of the plasma membrane of C. albicans. As seen in the result, the membrane disruptive action of dioscin caused morphological change and rhodamine leakage of the GUVs. In three-dimensional contour-plot analysis using flow cytometry, we observed a decrease in cell size, which is in agreement with our result from the GUV assay. These results suggest that dioscin exerts a considerable antifungal activity by disrupting the structure in membrane after invading into the fungal membrane, resulting in fungal cell death.

Enhancement of L-ornithine production by disruption of three genes encoding putative oxidoreductases in Corynebacterium glutamicum.

Recently, Corynebacterium glutamicum has been shown to exhibit gluconate bypass activity, with two key enzymes, glucose dehydrogenase (GDH) and gluconate kinase, that provides an alternate route to 6-phosphogluconate formation. In this study, gene disruption analysis was used to examine possible metabolic functions of three proteins encoded by open reading frames having significant sequence similarity to GDH of Bacillus subtilis. Chromosomal in-frame deletion of three genes (NCgl0281, NCgl2582, and NCgl2053) encoding putative NADP⁺-dependent oxidoreductases led to the absence of GDH activity and correlated with increased specific glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. This finding suggested that enhanced carbon flux from glucose was directed toward the oxidative pentose phosphate (PP) pathway, when the mutant was cultivated with 6 % glucose. Consequently, the mutant showed 72.4 % increased intracellular NADPH and 66.3 % increased extracellular L-ornithine production. The enhanced activities of the oxidative PP pathway in the mutant explain both the increased intracellular NADPH and the high extracellular concentration of L-ornithine. Thus, the observed metabolic changes in this work corroborate the importance of NADPH in L-ornithine production from C. glutamicum.

Prognostic factors for clinical outcomes after arthroscopic treatment of traumatic central tears of the triangular fibrocartilage complex.

Aims: The study aimed to assess the clinical outcomes of arthroscopic debridement and partial excision in patients with traumatic central tears of the triangular fibrocartilage complex (TFCC), and to identify prognostic factors associated with unfavourable clinical outcomes. Methods: A retrospective analysis was conducted on patients arthroscopically diagnosed with Palmer 1 A lesions who underwent arthroscopic debridement and partial excision from March 2009 to February 2021, with a minimum follow-up of 24 months. Patients were assessed using the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire, Mayo Wrist Score (MWS), and visual analogue scale (VAS) for pain. The poor outcome group was defined as patients whose preoperative and last follow-up clinical score difference was less than the minimal clinically important difference of the DASH score (10.83). Baseline characteristics, arthroscopic findings, and radiological factors (ulnar variance, MRI, or arthrography) were evaluated to predict poor clinical outcomes. Results: A total of 114 patients were enrolled in this study, with a mean follow-up period of 29.8 months (SD 14.4). The mean DASH score improved from 36.5 (SD 21.5) to 16.7 (SD 14.3), the mean MWS from 59.7 (SD 17.9) to 79.3 (SD 14.3), and the mean VAS pain score improved from 5.9 (SD 1.8) to 2.2 (SD 2.0) at the last follow-up (all p < 0.001). Among the 114 patients, 16 (14%) experienced poor clinical outcomes and ten (8.8%) required secondary ulnar shortening osteotomy. Positive ulnar variance was the only factor significantly associated with poor clinical outcomes (p < 0.001). Positive ulnar variance was present in 38 patients (33%); among them, eight patients (21%) required additional operations. Conclusion: Arthroscopic debridement alone appears to be an effective and safe initial treatment for patients with traumatic central TFCC tears. The presence of positive ulnar variance was associated with poor clinical outcomes, but close observation after arthroscopic debridement is more likely to be recommended than ulnar shortening osteotomy as a primary treatment.

Antifungal property of dihydrodehydrodiconiferyl alcohol 9'-O-beta-D-glucoside and its pore-forming action in plasma membrane of Candida albicans.

The aims of this study wereto investigate the antifungal activity as a bioactive property of dihydrodehydro-diconiferyl alcohol 9'-O-3-D-glucoside (DDDC9G) and the mode of action(s) involved in its effect. Antifungal susceptibility testing showed that DDDC9G possessed potent antifungal activities toward various fungal strains with almost no hemolytic effect. To understand the antifungal mechanism(s) of DDDC9G, we conducted the following experiments in this study using Candida albicans. Fluorescence experiments using the probe 1,6-eiphenyl-1, 3, 5-hexatriene (DPH) and propidium iodide suggested that DDDC9G perturbed the fungal plasma membrane. Consecutively, the analysis of the transmembrane electrical potential (DeltaPsi) with 3, 3'-dipropylthiadicarbocyanine iodide [DiSC3(5)] and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] indicated that DDDC9G induced membrane-depolarization. Furthermore, model membrane studies were performed wiith rhodamine-labeled giant unilamellar vesicles (GUVs), calcein encapsulating large unilamellar vesicles (ILUVs), and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of DDDC9G upon the fungal plasma membrane were through the formation of pores with the radii between 0.74 nm and 1.4 nm. Finally, in three dimensional (3D) flow cytometric contour plots, a reduced cell size was observed as a result of osmolarity changes from DDDC9G-induced structural and functional membrane damages.Therefore, the present study suggests that DDDC9G exerts its antifungal effect by damaging the membrane through pore formation in the fungal plasma membrane.

Prediction of an intracompartmental septum and its effect on outcomes of endoscopic release for de Quervain's syndrome.

LEVEL OF EVIDENCE: IV.

Radial versus dorsal plating for trapeziometacarpal arthrodesis: a comparative biomechanical study.

Trapeziometacarpal arthrodesis is used for the treatment of advanced arthritis. Insufficient stabilization of the joint may lead to nonunion or hardware problems after arthrodesis. The purpose of this study was to compare the biomechanical properties of dorsal versus radial plate fixation of the trapeziometacarpal joint in ten pairs of fresh-frozen cadaveric hands. The biomechanical performance of each group was measured for stiffness in extension and flexion and load to failure using cantilever bending tests. The stiffness in extension was lower in the dorsally positioned group than in the radially positioned group (12.1 versus 15.2 N/mm, respectively). Load to failure was comparable between both groups (53.9 versus 50.9 N, respectively). A radially positioned locking plate for trapeziometacarpal arthrodesis may be biomechanically advantageous.

The antimicrobial peptide arenicin-1 promotes generation of reactive oxygen species and induction of apoptosis.

BACKGROUND: Arenicin-1, a 21-residue antimicrobial peptide, is known to exert significant broad-spectrum antimicrobial activity without cytotoxicity in mammalian cells except at high concentration. However, the mechanism of fungal cell death by arenicin-1 is weakly understood. METHODS: We confirmed an increase in reactive oxygen species (ROS) in Candida albicans exposed to arenicin-1 and investigated the apoptotic response to ROS accumulation using apoptosis detecting methods. RESULTS AND CONCLUSIONS: Cells exposed to arenicin-1 showed an increase in the production of ROS and cytotoxic hydroxyl radicals, which are the major factors of apoptosis. The increase in ROS was due to mitochondrial dysfunction caused by arenicin-1. We confirmed that arenicin-1 induced mitochondrial membrane depolarization and also triggered release of activated metacaspases. Further, it initiated an apoptotic mechanism acting on the plasma membrane, including plasma membrane depolarization and exposure of phosphatidylserine on the outer surface. Cells finally died, showing morphological changes in the nucleus and DNA structure. Based on these apoptotic phenomena induced by arenicin-1, we concluded that arenicin-1 exerts antifungal activity by inducing apoptosis. GENERAL SIGNIFICANCE: This study suggests that the antimicrobial peptide arenicin-1 induces apoptosis in C. albicans via intracellular ROS accumulation and mitochondrial damage, resulting in fungal cell death.

Effects of positively charged arginine residues on membrane pore forming activity of Rev-NIS peptide in bacterial cells.

Here, we investigated antibacterial effects of Rev-NIS and suggested the role of positively charged amino acids on membrane pore forming activity of the peptide in bacterial cells, by synthesizing two analogs, Anal R and Anal S. Based on the amphipathic property of Rev-NIS, Anal R and Anal S were designed by substituting E(1) and L(3) to R and L(3) to S, respectively. The circular dichroism (CD) spectroscopy showed that Anal R and Anal S have the same conformation of Rev-NIS, with a significant fraction of helical structure. In succession, the antibacterial susceptibility testing showed that Rev-NIS and its analogs possessed significant activities (Anal R>Rev-NIS>Anal S), without hemolytic effects, against bacterial pathogens including antibiotics-resistant strains. Moreover, the membrane studies, 3,3'-dipropylthiadicarbocyanine iodide (diSC(3)5) staining and FITC-dextran (FD) leakage assay demonstrated that the analogs as well as Rev-NIS acted on the bacterial membranes and potently made pores, with the hydrodynamic radius between 1.4nm and 2.3nm. Especially, Anal R made larger pores than other peptides, with the radius between 2.3nm and 3.3nm. These results also corresponded to the result of antibacterial susceptibility testing. In summary, this study indicates that the two arginine residues are more influential than the hydrophobicity or the helicity, regarding the molecular activity of the peptide, and finally suggests that Anal R peptide may be applied to novel antibacterial agents.

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities.

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5'-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure-activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.