In situ observation of a cell adhesion and metabolism using surface infrared spectroscopy

In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the sample chamber of a FT-IR spectrometer was maintained at 37 °C and a humidified gas mixture containing 5% CO₂ was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7 cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm⁻¹) and also by the absorption intensities of CH _x bands (2,700–3,100 cm⁻¹). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method based on infrared absorption spectroscopy has a potential for bioscreening application.     

NO-1886改善糖尿病小型猪的糖代谢

合成化合物NO-1886是一种脂蛋白脂酶活化剂,已被证明其可降低血浆TG并能升高HDLC的浓度．后又发现其还有降低高脂高蔗糖诱发糖尿病兔血浆葡萄糖浓度的作用．对高脂高蔗糖饲料喂养的小型猪脂肪细胞大小、血浆TNF—α和FFA的水平以及NO-1886对其影响进行了研究,结果发现,脂肪细胞明显肥大．血浆TNF-α和FFA以及空腹血糖水平均增高,且引起胰岛素抵抗．添加了l％NO-1886后．脂肪细胞增大被抑制,血浆TNF—α、FFA和空腹血糖的浓度均显著降低,血浆葡萄糖清除率和胰岛素分泌急性相都有了明显改善．以上结果说明,NO-1886可能通过抑制脂肪蓄积、降低血浆TNF-α和FFA的浓度而改善高脂高蔗糖饲料引起的小型猪的糖代谢紊乱．     

Chromosomal Mapping of Genes in theRBCS Family in Arabidopsis thaliana

In Arabidopsis thaliana, four genes have been identified inthe RBCS gene family, one being assigned to subfamily RBCS-Aand the other three to subfamily RBCS-B (1B, 2B and 3B). Todetermine the chromosomal location ofthese genes, hybridizationanalysis with CIC YAC high-density filters was carried out forthe RBCS-A gene, and CAPS analysis for the three RBCS-B genes,based on the finding that restriction fragment length polymorphismis present in the upstream region of the gene RBCS-3B. The RBCS-Agene was mapped at 100.8 cM from the top of chromosome 1 andthe three RBCS-B genes at 62.70 cM from the top of chromosome5.     

NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.     

Higher serum bone alkaline phosphatase as a predictor of mortality in male hemodialysis patients

AimsHigher serum alkaline phosphatase predicts lower mortality in chronic kidney disease and hemodialysis patients without liver dysfunction because it reflects high bone turnover. The purpose of our study was to compare the significance of serum bone alkaline phosphatase (BAP) with that of other bone markers in prediction of all-cause mortality(ACM) in male hemodialysis patients.Main methodsThe study was performed for 5 years. Serum BAP, intact osteocalcin (iOC), ß-CrossLaps (CTX), and intact parathyroid hormone (iPTH) were measured in 196 male hemodialysis patients without radiographic fracture. Their day-to-day variation during 5 consecutive days and diurnal variation were determined in 13 healthy males.Key findingsThe patients were divided into higher and lower groups based on serum levels of bone markers(mean ± SD: iPTH 218.6 ± 214.5 pg/ml, BAP 23.6 ± 12.2U/L, iOC 42.8 ± 45.2 ng/ml, CTX 1.71 ± 1.23 nmol/L BCE). In Kaplan–Meier analysis, the higher BAP group had significantly higher ACM than the lower BAP group (P = 0.013), whereas mortality did not differ between the higher and lower groups in other markers. Cox regression hazard analysis identified higher log BAP as a significant independent predictor [hazard ratio(HR) 8.32(95%CI:1.18–58.98)] for ACM after adjustment for various factors including pre-existing cardiovasucular disease, presence of DM. The significant association of mortality with serum BAP alone, in contrast with other markers including CTX [HR0.64 (95%CI:0.16–2.47)], iOC [HR0.97(95%CI:0.36–2.64)], iPTH [HR0.84(95%CI:0.44–1.60)],it may be due to the narrower day-to-day variation and the absence of diurnal variation in serum BAP compared to other markers.SignificanceHigher serum BAP may be a predictor of ACM in male hemodialysis patients.     

Long-term feeding on powdered food causes hyperglycemia and signs of systemic illness in mice

Aims

Dietary habits are crucial factors affecting metabolic homeostasis. However, few animal experiments have addressed the effects of long-term feeding with soft food on parameters reflecting systemic health.

Main methods

Using mice, we compared the effects of short (3 days) and long (17 weeks from weaning) feeding periods between powdered food and normal pellet food on the levels of blood glucose, serum levels of insulin, catecholamines, and corticosterone, blood pressure, and/or social interaction behaviors. In addition, the effects of a human glucagon-like peptide-1 analog, liraglutide (a new drug with protective effects against neuronal and cardiovascular diseases), were compared between the powder and pellet groups.

Key finding

(i) Powdered food, even for such a short period, resulted in a greater glycemic response than pellet food, consistent with powdered food being more easily digested and absorbed. (ii) Long-term feeding on powdered food induced hyperglycemia and related systemic signs of illness, including increases in serum adrenaline, noradrenaline, and corticosterone, higher blood pressures (especially diastolic), and increased social interaction behaviors. (iii) Liraglutide, when administered subcutaneously for the last 2 weeks of the 17-week period of feeding, improved these changes (including those in social interaction behaviors).

Significance

The hyperglycemia associated with long-term powdered-food feeding may lead to certain systemic illness signs, such as elevations of blood glucose, hypertension, and abnormal behaviors in mice. Mastication of food of adequate hardness may be very important for the maintenance of systemic (physical and mental) health, possibly via reduction in the levels of blood glucose and/or adrenal stress hormones (catecholamines and glucocorticoids).     

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.     

Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.     

Single nucleotide polymorphisms of thrifty genes for energy metabolism: evolutionary origins and prospects for intervention to prevent obesity-related diseases

The "thrifty" genotype and phenotype that save energy are detrimental to the health of people living in affluent societies. Individual differences in energy metabolism are caused primarily by single nucleotide polymorphisms (SNPs), some of which promote the development of obesity/type 2 diabetes mellitus. In this review, four major questions are addressed: (1) Why did regional differences in energy metabolism develop during evolution? (2) How do genes respond to starvation and affluence? (3) Which SNPs correspond to the hypothetical "thrifty genes"? (4) How can we cope with disease susceptibility caused by the "thrifty" SNPs? We examined mtDNA and genes for energy metabolism in people who live in several parts of Asia and the Pacific islands. We included 14 genes, and the SNP frequencies of PPAR gamma 2, LEPR, and UCP3-p and some other genes differ significantly between Mongoloids and Caucasoids. These differences in SNPs may have been caused by natural selection depending on the types of agriculture practiced in different regions. Interventions to counteract the adverse effects of "thrifty" SNPs have been partially effective.     

Structural properties of mammalian mitochondrial ADP/ATP carriers: identification of possible amino acids that determine functional differences in its isoforms

The structural properties of isoforms of the mitochondrial ADP/ATP carrier expressed in mammals were characterized in order to understand their possible functional differences. To accomplish this, the cDNA clone of the bovine type 2 isoform was isolated and characterized. We also extensively explored the rat type 3 isoform, but it was not detected. We next compared the amino acid sequences of the ten ADP/ATP carriers, which are expressed in mammals. As a result, amino acids at positions 45, 147 and 164 were found to show strict isoform dependency regardless of species differences. Thus, they are expected to determine functional differences in the isoforms of the ADP/ATP carrier.