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To evaluate whether continuous subcutaneous insulin infusion attenuates the progression of diabetic complications, we retrospectively extracted data from 35 individuals who had developed type 1 diabetes mellitus aged ≤20 years and whose treatment had been changed from multiple daily injections to continuous subcutaneous insulin infusion. The annual changes in estimated glomerular filtration rate, urinary albumin excretion rate, carotid intima-media thickness and brachial-ankle pulse wave velocity during each treatment period were calculated. Although mean glycated hemoglobin under the continuous subcutaneous insulin infusion treatment was lower than that under the multiple daily injection treatment, there were no significant differences in annual changes in diabetic nephropathy and atherosclerosis between the two treatment periods. This pilot study showed that, in Japanese patients with juvenile-onset type 1 diabetes mellitus, there was no significant difference in the progression of diabetic nephropathy and atherosclerosis, at least in the early stage, between the two treatments.

Aims/Introduction Diabetes patients develop a variety of metabolic abnormalities in addition to hyperglycemia. However, details regarding change in various metabolites after comprehensive diabetes treatment remain unknown. This study aimed to identify the short‐term change in metabolome in inpatients who were subject to comprehensive diabetes treatment, using gas chromatography/mass spectrometry‐based non‐target metabolomics techniques. Materials and Methods Participants of the present study were randomly recruited from the patients with type 2 diabetes hospitalized due to problems with glycemic control (n = 31) and volunteers without diabetes (n = 30), both of whom were aged between 20 and 75 years. A metabolomic analysis of fasting plasma samples on the 2nd (pre‐treatment) and 16th hospital (post‐treatment) day with gas chromatography/mass spectrometry using a multiple reaction monitoring mode was carried out. Results A principal component analysis showed that metabolome of fasting plasma was different between individuals with and without diabetes. The metabolome of fasting plasma in diabetes patients after treatment was different from that of pre‐treatment, as well as individuals without diabetes. Many amino acids (proline, glycine, serine, threonine, methionine, pyroglutamic acid, glutamine and lysine) were significantly increased by >10% after administering the inpatient diabetes treatment. A hierarchical clustering analysis showed that in the case of patients with markedly decreased monosaccharide levels and increased 1,5‐anhydroglucitol, the levels of amino acids increased more significantly. Conclusions After a 2‐week comprehensive treatment, the plasma levels of various amino acids increased in conjunction with the reduction in monosaccharide levels in poorly controlled type 2 diabetes patients., This study aimed to identify the short‐term change in metabolome in inpatients who were subject to comprehensive diabetes treatment, using gas chromatography/mass spectrometry‐based non‐target metabolomics techniques. After a 2‐week comprehensive treatment, the plasma levels of various amino acids increased in conjunction with the reduction in monosaccharide levels in poorly controlled type 2 diabetes patients.

Recently, it is widely recognized that microinflammation plays important roles in the pathophysiology of metabolic diseases, especially obesity-related disorders, diabetes and their complications. Lipopolysaccharide-binding protein (LBP) is a liver-derived acute-phase protein responsive to lipopolysaccharides (LPS) produced by gram-negative bacteria, thus reflects the systemic inflammation caused by the infection of those bacteria including gut dysbiosis. In this study, we evaluated the plasma LBP levels and investigated its clinical significance in 67 Japanese patients with type 1 diabetes. Univariable analysis showed that LBP levels were significantly associated with body mass index (BMI; r = 0.43, p

Recent progress in research on glucagon and α‐cells highlights their pathophysiological roles in diabetes. We previously showed that plasma glucagon levels measured by newly developed enzyme‐linked immunosorbent assay were dysregulated in patients with type 1 diabetes with respect to plasma glucose levels, suggesting dysregulated secretion. In the current study, the annual change in plasma glucagon levels was assessed in these same patients. We found that the plasma glucagon levels in the 66 Japanese patients involved in the study were significantly correlated between both years. In addition, they were significantly associated with serum blood urea nitrogen levels in a multivariate linear regression analysis, as reported in our previous study. The statistical correlation in glucagon levels between annual checkups and the sustained significant correlation between glucagon and blood urea nitrogen suggest a constant dysregulation of glucagon in association with altered amino acid metabolism in patients with type 1 diabetes., The annual plasma glucagon levels in 66 Japanese patients with type 1 diabetes were significantly correlated between both years. The plasma glucagon levels were significantly associated with serum blood urea nitrogen levels. The results suggest constant dysregulation of glucagon in association with altered amino acid metabolism in patients with type 1 diabetes.

Pancreas transplantation (PTx) has been performed worldwide for patients with type 1 diabetes accompanied with end-stage renal disease or uncontrollable glycemic fluctuation. Nevertheless, risk factors of posttransplant glucose intolerance, which is responsible for progress of diabetic complications, remains unclear, especially in cases without pancreatic graft function loss. Therefore, this study was conducted to search for predictive factors of future glucose tolerance in PTx recipients without pancreatic graft function loss. Subjects were selected from among 41 Japanese patients with type 1 diabetes who received PTx between 2000 and 2016 in Osaka University Hospital, and 24 subjects free from rejections and thromboses were analyzed. Several examinations to evaluate insulin secretion and insulin sensitivity within 6 months after transplantation (initial examination) were performed. Glucose tolerance was evaluated by 120-minute post-load plasma glucose level during 75-g oral glucose tolerance tests (OGTT), referred to as PGOGTT120, at the initial examination and between 1 year and 2 years posttransplantation (maintenance period). The initial examination factors that were correlated with PGOGTT120 in the maintenance period were PGOGTT120 [r = 0.52 (p = 0.01)], insulinogenic index [r = -0.65 (p < 0.01)], and the ratio of incremental area under the curve of insulin to that of plasma glucose (iAUCR) calculated from data of OGTT [r = -0.65 (p < 0.01)]. Insulinogenic index [β = -0.28 (p = 0.02)] and iAUCR [β = -0.29 (p = 0.02)] were still significantly correlated with PGOGTT120 in the maintenance period after adjustment for PGOGTT120 at the initial examination. In conclusion, insulinogenic index and iAUCR from OGTT performed in the early posttransplantation period were predictive factors of future glucose intolerance.

The newly designed green fluorescent protein (GFP) chromophore analog, bar-DAIN, which has a 2-biaryl-conjugated 5-(diphenylmethylene)imidazolinone structure, was effectively synthesized using the Suzuki coupling reaction. Bar-DAIN showed environment-dependent fluorescence behavior; for example, the thienyl analog emitted yellow-green fluorescence in viscous solution (λem: 535 nm), yellow-orange fluorescence in suspension (λem: 551 nm), and cyan fluorescence in a powder state (λem: 497 nm) although it showed almost no emission in common solvents such as dichloromethane. The dynamic discoloration of the fluorescence was observed by changing environmental conditions from suspension to viscous.

A novel fluorescent cytidine derivative (dCDAIN) bearing 4-(diarylmethylene)imidazolinones (DAINs) at C5-position was successfully synthesized and incorporated into oligonucleotides. The oligonucleotide bearing dCDAIN hybridized with complimentary strand in a sequence specific manner, and the florescence intensity was changed depending on the nucleobases opposite to dCDAIN in the double stranded DNA or RNA. Interestingly, the fluorescence intensity was higher when the dCDAIN-containing oligonucleotide hybridized with a mismatched sequence. Especially, when dCDAIN was located at the opposite position of an abasic site, the fluorescent intensity was four times higher than that observed in a single stranded state.

Aims Tissue accumulatedadvanced glycation end products (AGEs) can be evaluated non-invasively by an autofluorescence reader as skin autofluorescence (skin AF)·The present study investigated whether skin AF is associated with diabetic micro- and macroangiopathies in Japanese patients with type 2 diabetes mellitus (T2DM). Methods Skin AF was measured in 193 enrolled Japanese patients with T2DM and 24 enrolled healthy non-diabetic subjects by using the AGE reader®. Diabetic micro- and macroangiopathies were evaluated in the T2DM patients. Results Skin AF was significantly increased in patients with T2DM than in age- and sex-matched non-diabetic controls (2.35 ± 0.51 [mean ± SD] and 1.91 ± 0.29, respectively, p = 0.001). In subjects with T2DM, skin AF was associated with age, pack-years of smoking, and eGFR (estimated glomerular filtration rate) independently. Skin AF was significantly increased in patients with diabetic retinopathy, neuropathy, nephropathy, and macroangiopathy than in those without them, and significantly associated with the number of diabetic complications. Moreover, skin AF was an independent predictor for diabetic retinopathy, neuropathy, and nephropathy but not macroangiopathy, after adjusting for major traditional risk factors. Conclusions Skin AF is an independent predictor for diabetic retinopathy, neuropathy and nephropathy in Japanese patients with T2DM.

We have developed a novel analog of the GFP chromophore: geo-DAIN. Since geo-DAIN is equipped with an E/Z-photoisomerizable geometrical diarylmethylene moiety instead of benzylidene of the GFP chromophore, different-colored reversible emissions are expected. We synthesized geo-DAIN by a condensation with methyl imidate and N-(diarylmethylene)glycinate. We found the emission from geo-DAIN to be different from that of benzylidene-type analogs; in the powder state, the E- and Z-isomers of geo-DAIN emitted different fluorescence colors.

Currently, the clinical dynamics of glucagon need to be revised based on previous data obtained from conventional glucagon radioimmunoassays. In the present study, we evaluated plasma glucagon levels in type 1 diabetes patients using a newly‐developed sandwich enzyme‐linked immunosorbent assay, and its association with clinical parameters and markers of diabetes complications were statistically assessed. The plasma glucagon level in 77 Japanese type 1 diabetes patients was 28.1 ± 17.7 pg/mL, and comparable with that reported previously for type 2 diabetes patients. However, the values were widely spread and did not correlate with plasma glucose values. Additionally, the average glucagon levels in patients in a hypoglycemic state (glucose level

Diarylmethylenated and cholestene (or -tane)-hybrid analogues of the GFP chromophore, namely, Ch-DAINs were successfully synthesised by a condensation reaction between methyl imidates and N-(diarylmethylene)glycinates. Among the Ch-DAINs synthesised, a diphenyl-type analogue showed viscosity-dependent and cholesterol-responsive fluorescent properties. It showed a nearly linear increase of the fluorescence emission in triglycerides and vesicles as the amount of cholesterol was increased.

Pdx1, a β-cell-specific transcription factor, has been shown to play a crucial role in maintaining β-cell function through transactivation of β-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against β-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in β cells (βPdx1) and crossed these mice with Ins2Akita diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2Akita mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of βPdx1; Ins2Akita mice. The βPdx1; Ins2Akita mice exhibited significantly improved glucose tolerance, compared with control Ins2Akita littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that βPdx1; Ins2Akita mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of βPdx1; Ins2Akita mice. These findings suggest that the sustained expression of Pdx1 improves β-cell failure in Ins2Akita mice, at least partially through the preserving expression of β-cell-specific genes as well as improved localization of GLUT2.

The objective of the present study was to investigate the correlations between serum under-carboxylated osteocalcin (ucOC) or osteocalcin (OC) concentrations and %body fat, serum adiponectin and free-testosterone concentration, muscle strength and dose of exogenous insulin in patients with type 1 diabetes. We recruited 73 Japanese young adult patients with childhood-onset type 1 diabetes. All participants were receiving insulin replacement therapy. The correlations between logarithmic serum ucOC or OC concentrations and each parameter were examined. Serum ucOC and OC concentrations were inversely correlated with% body fat (r = -0.319, P = 0.007; r = -0.321, P = 0.006, respectively). Furthermore, multiple linear regression analyses were performed to determine whether or not serum ucOC or OC concentrations were factors associated with %body fat. Serum ucOC and OC concentrations remained significant factors even after adjusting for gender, HbA1c, body weight-adjusted total daily dose of insulin and duration of diabetes (β = -0.260, P = 0.027; β = -0.254, P = 0.031, respectively). However, serum ucOC and OC concentrations were not correlated with serum adiponectin or free-testosterone concentrations, muscle strength or dose of exogenous insulin. In conclusion, our study demonstrates the inverse correlation between serum ucOC or OC concentrations and body fat in patients with type 1 diabetes.

Aims/Introduction The aim of the present study was to examine the short- and long-term effect of sitagliptin on glucose tolerance after near normalization of glycemic control with insulin in poorly controlled type 2 diabetic patients. Materials and Methods We consecutively enrolled a total of 30 type 2 diabetic patients whose glycated hemoglobin levels (National Glycohemoglobin Standardization Program) were ≥7.4%, stopped all oral antidiabetic drugs and started insulin therapy. When fasting plasma glucose levels became

The aim of this study was to examine the possible association of vitamin D deficiency with diabetic retinopathy in 75 young Japanese type 1 diabetic patients. A multivariate regression analysis, duration of diabetes and vitamin D deficiency were independent determinants of diabetic retinopathy.

Novel dendrimers having an adamantane structure as a core were synthesized such that even low generation dendrimers had a globular structure. Moreover we tried to give them biodegradable function by using ester bonds. Synthesis of the dendrimers, particularly at higher generations, proved difficult via a stepwise procedure, and thus a convergent route was used in which the adamantane core is coupled to the dendritic segments in the final step. We achieved the synthesis of two separate dendrimers with convergent methods till the third generations. The convergent dendrimers were synthesized in good yields compared with divergent one and both dendrimers were found to have narrow polydispersities by GPC analysis.

It is still controversial whether circulating soluble form of receptor for AGE (sRAGE) is associated with atherosclerosis in diabetic patients. In this study, we enrolled 276 Japanese type 2 diabetic subjects without history of cardiovascular disease (CVD), assessed their baseline clinical and biochemical data including serum sRAGE levels, and prospectively evaluated the association between these parameters and CVD events. The median follow-up period was 5.6 years and there were 25 new CVD events. The tertile analysis showed that the risk for CVD events was higher as serum sRAGE levels were increased (p for trend = 0.046). A multivariate Cox proportional hazards regression analysis revealed that serum sRAGE levels were independently associated with CVD (HR per 1SD = 1.59, 95% CI 1.04-2.45, p = 0.034), even after adjusting for conventional coronary risk factors. In summary, elevated sRAGE levels were associated with the increased risk of CVD in Japanese type 2 diabetic subjects.

YKL-40 is a marker of inflammation and endothelial dysfunction, both of which play important roles in the progression of diabetic complications. However, little information has been obtained about serum YKL-40 levels in type 1 diabetic patients. We evaluated YKL-40 levels and its association with diabetic micro- and macroandgiopathy in 131 young Japanese type 1 diabetic patients without advanced diabetic complications (aged 24.7±5.9 years) and 97 age- and gender-matched healthy controls. YKL-40 levels were significantly elevated in type 1 diabetic patients than in healthy controls (median (range) 46.4 (20.3-136.7) and 52.3 (21.4-274.1) ng/mL, respectively, p = 0.003). There was a significant positive association between YKL-40 levels and urinary albumin creatinine ratio (UACR) (r = 0.226, p = 0.013). Furthermore, a multivariate regression analysis demonstrated that YKL-40 levels were a determinant of UACR independently of conventional risk factors. In addition, YKL-40 levels were significantly higher in participants with diabetic retinopathy compared to those without it (median (range) 55.5 (23.3-274.1) and 50.3 (21.4-237.4) ng/mL, respectively, p = 0.039). Serum YKL-40 levels were elevated in type 1 diabetic patients and associated with increasing level of albuminuria. YKL-40 could be a predictor to assess the risk of diabetic microangiopathy in the early stage in type 1 diabetic patients.

We herein report the case of a 41-year-old Japanese female office worker who developed symptomatic hypocalcemia with severe vitamin D deficiency following treatment for Graves’ disease with methimazole. The patient’s hypocalcemia was mainly caused by vitamin D deficiency due to unbalanced diets and inadequate exposure to sunlight in addition to the resolution of hyperthyroidism. Vitamin D deficiency is increasing worldwide, and it has been more recently shown to relate to the pathogenesis of Graves’ disease. However, vitamin D deficiency as a cause of hypocalcemia has received little attention. Taken together, this case suggests that we should take more care in calcium kinetics and vitamin D status during treatment for Graves’ disease with antithyroid drugs.

A small molecular model compound for the green fluorescent protein chromophore was readily synthesized by a novel condensation reaction of (thio)imidate with imino-ester via an aziridine intermediate. This compound showed fluorescence in the solid and frozen solution states but not in the solution state. Its fluorescent property was successfully applied in the detection of dsDNA.

Epoxypiperidine derivatives bearing a dehydroamino acid unit were designed and synthesized as novel DNA alkylating agents based on the structure of azinomycins. A relaxation assay of plasmid DNA revealed that the epoxypiperidine derivative 3 has a DNA cleavage activity. Based on the studies, it would appear that the electron density of the amino group of epoxypiperidines plays a critically important role in the DNA cleavage.

We designed a novel 2′- O ,5′- N bridged nucleic acid, 2′,5′-BNA ON , whose sugar puckering was fixed to S-type conformation by an N – O linkage. A dimer unit formed from 2′,5′-BNA ON -U and thymidine was synthesized via a coupling reaction between a protected 2′,5′-BNA ON -U monomer and a thymidine derivative. Introduction of 2′,5′-BNA ON -U into DNA was carried out using conventional phosphoramidite chemistry with a DNA synthesizer. The hybridization abilities of 2′,5′-BNA ON -U-modified oligonucleotides against DNA or RNA complement were evaluated.

Leustroducsin B was synthesized via a convergent route based on division of the leustroducsin molecule into three segments A, B, and C. Two coupling reactions (Julia coupling reaction and Nozaki-Hiyama-Kishi (NHK) reaction) were employed for coupling of segments A and B: segment A1 for the Julia coupling reaction was prepared by a combination of Sharpless asymmetric epoxidation and an epoxide-cleavage reaction with an organoaluminum reagent, while segment A2 for the NHK reaction was synthesized from optically active alcohol that had previously been prepared by lipase-catalyzed kinetic resolution. Segment B, whose structure was modified with some functional groups, was synthesized from (R)-malic acid by a combination of Wittig reaction and Sharpless asymmetric dihydroxylation, and segment C, containing a cyclohexane moiety, was prepared by asymmetric Diels-Alder reaction. Segment B was first coupled with segment A1 via the Julia coupling reaction, but the yield was low due to unexpected epimerization. The NHK reaction of segment A2 proceeded to give the coupling product in good yield. This product was coupled with segment C via Wittig and Stille coupling reactions, and finally, phosphorylation was carried out by partial hydrolysis of a cyclic phosphate to give leustroducsin B.

Total synthesis of leustroducsin B was achieved via a convergent route, which includes Julia coupling reaction of segment A with segment B followed by Stille coupling reaction of segment C.

Fostriecin (CI-920), isolated from Streptomyces pulveraceus, shows in vitro antitumor activity against leukemia, lung breast and ovarian cancer, and in vivo against L 1210 and P 388 leukemia. Its analogous compounds, such as leustroducsins, phoslactomycins and cytostatin, are also known to show a variety of biological activities. One of the most characteristic biochemical features is a specific inhibitory activity against Ser/Thr protein phosphatase 2 A. Not only because of their interesting biological activities, but also in order to prove utility of new reactions and/or new synthetic methodologies, fostriecin and its analogs attract much attention as a synthetic target, and a number of synthetic studies on these compounds have been conducted, particularly since the stereostructure of fostriecin was revealed by Boger's group in 1997. In this review, we focus on the studies reported after Shibasaki's review in 2005. Recent synthetic studies on the analogous natural products, leustroducsins, phoslactomycins and cytostatin are also included.

The novel condensation reaction between imidates or thioimidates with N-(diarylmethylene)glycinates affords imidazolinone derivatives with interesting fluorescence properties.

A novel and highly efficient procedure for inside selective esterification of terminal vic -diols has been achieved in one-pot via Yb(OTf) 3 -catalyzed formation and partial hydrolysis of cyclic orthoesters. This method offers several advantages including wide compatibility with acid labile functional groups, and good to high regioselectivity and yields.

The total synthesis of an antitumor antibiotic, fostriecin (CI-920), via a highly convergent route is described. A characteristic feature of the present total synthesis is that the synthesis was achieved via a coupling procedure of three segments A, B, and C. The unsaturated lactone moiety of fostriecin, corresponding to segment A, was constructed from a known Horner-Emmons reagent, and the stereochemistry of the C-5 position was introduced by asymmetric reduction with (R)-BINAl-H. Segment B having a series of stereogenic centers was synthesized from (R)-malic acid and the stereogenic centers at the C-8 and C-9 positions were prepared by a combination of Wittig reaction and Sharpless asymmetric dihydroxylation reaction. The conjugated Z,Z,E-triene moiety of fostriecin, corresponding to segment C, was eventually constructed by Wittig reaction and Stille coupling reaction. The phosphate moiety, which is known to be essentially important for the antitumor activity, was introduced via two routes: (i) direct phosphorylation of the monohydroxyl derivative in which other hydroxyl groups are protected with silyl groups; (ii) cyclic phosphorylation and selective cleavage of the cyclic phosphate derivative. Although the former route is basically the same as those reported by other groups, the latter route is novel and more effective than the former one. The present total synthesis would serve as a versatile synthetic route to not only fostriecin, but also its various analogues including stereoisomers.

Catalytic transformation of serine- O -carbonate to S -aryl cysteine derivatives was successfully achieved in the presence of Li + by the use of a pyridoxal model compound having an ionophore function, which is the first example mimicking cystathionine β-synthase, artificially.

As a second-generation pyridoxal model compound for cystathionine β-synthase, we designed a novel model compound having an ionophore function and an imidazole function, application of which to the β-replacement reaction with various thiols smoothly took place to give S-substituted cysteines. Peptides having a serine-O-carbonate residue at the N-terminal position were also converted to the corresponding peptides having an S-substituted cysteine residue under the catalytic conditions of the novel pyridoxal model compound.

Graves’ disease (GD) is an autoimmune thyroid disease in which thyrotropin receptor autoantibodies (TRAb) cause hyperthyroidism. Although medical treatment with antithyroid drugs (ATD) is the first choice treatment for GD in Japan and Europe, a remission rate of GD with ATD is not satisfactory, and many patients need long-term treatment with ATD or further treatments such as radioactive iodine therapy or thyroidectomy [1]. Therefore, it is very important to identify the factors relating to the remission of GD. It has been recently shown that vitamin D deficiency is associated with the onset and/or development of several autoimmune diseases, including multiple sclerosis (MS), inflammatory bowel disease (IBD), and type 1 diabetes (T1DM) [2]. Furthermore, it has been reported more recently that patients with autoimmune thyroid diseases including GD have lower vitamin D status [3, 4]. However, there is no study comparing vitamin D status between the patients with and without remission of GD. In the present study, we examined vitamin D status in female patients with and without remission of GD and discussed the role of vitamin D in the pathogenesis and/or prognosis of GD.

Improvement of SiO2/Si interface quality and its effect on the performance of low-temperature-processed polycrystalline silicon thin-film transistors (poly-Si TFTs) are investigated. Two gate SiO2 formation conditions for realizing the density of interface states (Dit) at mid-gap of 1.4×1011 and 4.1×1010 cm-2eV-1 were applied to a 425°C TFT fabrication process using electron cyclotron resonance (ECR) plasma enhanced chemical vapor deposition (PECVD). By reducing Dit, reduction of threshold voltage from 1.97 to 1.12 V, reduction of sub-threshold swing from 303 to 250 mV/decade and increase of mobility from 196 to 309 cm2V-1s-1 were observed. The analysis of TFT characteristics indicated the decrease of both deep and shallow level trap states. As a result, not only threshold voltage and sub-threshold swing, but also the mobility of the poly-Si TFT was significantly improved. The results indicate that low-temperature process technologies for forming a high-quality SiO2/Si interface are important for next-generation high-performance poly-Si TFTs.

We examined a possible association between subclinical hypothyroidism and albuminuria in 159 people with type 2 diabetes. Patients with subclinical hypothyroidism had significantly higher levels of urinary albumin-to-creatinine ratio (UACR) than those with euthyroidism. Multivariate logistic regression analyses demonstrated that serum TSH level was an independent risk factor of albuminuria.

While the exogenous expression of a combination of transcription factors have been shown to induce the conversion of non-β cells into insulin-producing cells, the reprogramming efficiency remains still low. In order to develop an in vitro screening system for an optimized reprogramming protocol, we generated the reporter cell line mPac-MIP-RFP in which the reprogramming efficiency can be quantified with red fluorescent protein expressed under the control of the insulin promoter. Analysis with mPac-MIP-RFP cells sequentially infected with adenoviruses expressing Pdx1, Neurog3, and Mafa revealed that expression of Pdx1 prior to Neurog3 or Mafa augments the reprogramming efficiency. Next, infection with a polycistronic adenoviral vector expressing Pdx1, Neurog3 and Mafa significantly increased the expression level of insulin compared with the simultaneous infection of three adenoviruses carrying each transcription factor, although excessive expression of Mafa together with the polycistronic vector dramatically inhibited the reprogramming into insulin-producing cells. Thus, in vitro screening with the mPac-MIP-RFP reporter cell line demonstrated that the timing and dosage of gene delivery with defined transcription factors influence the reprogramming efficiency. Further investigation should optimize the reprogramming conditions for the future cell therapy of diabetes.

Regio- and stereoselective α-alkylation of N-terminal amino acid residue of peptides was achieved by Li+-mediated alkylation of aldimines prepared from the peptides and a pyridoxal model compound having a chiral ansa-structure and an ethoxyethoxy group at C-3. The stereochemistry and stereoselectivity of the reaction were found to be influenced predominantly by the chirality of the model compound and Li+, but little by the stereochemistry of the original peptides.