Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.     

Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract

Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha.     

Real-time observation of conformational fluctuations in Zn-substituted myoglobin by time-resolved transient hole-burning spectroscopy.

Equilibrium fluctuations of the protein conformation have been studied in myoglobin by a novel method of time-resolved transient hole-burning spectroscopy over a temperature range of 180-300 K and a time range of 10 ns to 10 ms. The temporal shift of the hole spectrum has been observed in a wide temperature region of 200-300 K. It has been found that the time behavior of the peak position of the hole is highly nonexponential and can be expressed by a stretched exponential function with a beta value of 0.22. As compared with the results for a dye solution sample, the time scale of the fluctuation of the protein conformation is much more weakly dependent on temperature. The time scale of the observed conformational dynamics shows a temperature dependence similar to that associated with the ligand escape process of myoglobin.     

Risk Score to Predict 1-Year Mortality after Haemodialysis Initiation in Patients with Stage 5 Chronic Kidney Disease under Predialysis Nephrology Care

Background

Few risk scores are available for predicting mortality in chronic kidney disease (CKD) patients undergoing predialysis nephrology care. Here, we developed a risk score using predialysis nephrology practice data to predict 1-year mortality following the initiation of haemodialysis (HD) for CKD patients.

Methods

This was a multicenter cohort study involving CKD patients who started HD between April 2006 and March 2011 at 21 institutions with nephrology care services. Patients who had not received predialysis nephrology care at an estimated glomerular filtration rate (eGFR) of approximately 10 mL/min per 1.73 m² were excluded. Twenty-nine candidate predictors were selected, and the final model for 1-year mortality was developed via multivariate logistic regression and was internally validated by a bootstrapping technique.

Results

A total of 688 patients were enrolled, and 62 (9.0%) patients died within one year of HD initiation. The following variables were retained in the final model: eGFR, serum albumin, calcium, Charlson Comorbidity Index excluding diabetes and renal disease (modified CCI), performance status (PS), and usage of erythropoiesis-stimulating agent (ESA). Their β-coefficients were transformed into integer scores: three points were assigned to modified CCI≥3 and PS 3–4; two to calcium>8.5 mg/dL, modified CCI 1–2, and no use of ESA; and one to albumin<3.5 g/dL, eGFR>7 mL/min per 1.73 m², and PS 1–2. Predicted 1-year mortality risk was 2.5% (score 0–4), 5.5% (score 5–6), 15.2% (score 7–8), and 28.9% (score 9–12). The area under the receiver operating characteristic curve was 0.83 (95% confidence interval, 0.79–0.89).

Conclusions

We developed a simple 6-item risk score predicting 1-year mortality after the initiation of HD that might help nephrologists make a shared decision with patients and families regarding the initiation of HD.     

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 10³ cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.     

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.     

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

Background and aims

The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.

Methods

Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.

Results

We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.

Conclusions

The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.     

Role of oxidative stress in vinorelbine-induced vascular endothelial cell injury

Vinorelbine (VNR), a vinca alkaloid anticancer drug, often causes vascular injury such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis. The purpose of this study was to identify the mechanisms that mediate the cell injury induced by VNR in porcine aorta endothelial cells (PAECs). PAECs were exposed to VNR for 10 min followed by further incubation in serum-free medium without VNR. The exposure to VNR (0.3–30 μM) decreased the cell viability concentration and time dependently. The incidence of apoptotic cells significantly increased at 12 h after transient exposure to VNR. At the same time, VNR increased the activity of caspases. Interestingly, VNR rapidly depleted intracellular glutathione (GSH) and increased intracellular reactive oxygen species (ROS) production. Moreover, VNR depolarized the mitochondrial membrane potential and decreased cellular ATP levels. These VNR-induced cell abnormalities were almost completely inhibited by GSH and N-acetylcysteine. On the other hand, l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, aggravated the VNR-induced loss of cell viability. These results clearly demonstrate that VNR induces oxidative stress by depleting intracellular GSH and increasing ROS production in PAECs, and oxidative stress plays an important role in the VNR-induced cell injury.     

Exhaustive crystal structure search and crystal modeling of beta-chitin

An exhaustive search of the crystal structure of beta-chitin was carried out by simultaneously optimizing all the structural parameters based on published X-ray diffraction data and stereochemical criteria. The most probable structure was characterized by a parallel-up chain polarity, a gg orientation of hydroxymethyl groups and an intermolecular hydrogen bond along the a-axis, which essentially reproduced the original structure proposed by Gardner and Blackwell. The proposed crystal structure was subsequently subjected to crystal modeling using the AMBER force field. The probable orientation of hydroxyl groups and their motional behaviors is proposed based on calculations for the crystal models identified. Solvated crystal models exhibited a slightly deformed structure with the formation of appreciable numbers of hydrogen bonds along the b-axis.