Iron fortification of rice seeds through activation of the nicotianamine synthase gene

The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression.     

Assessment of compliance for oral medicines with MMSE, Mini-Mental State Examination, in hospitalized elderly patients

The minimental state examination (MMSE) is a widely used, standardized method to assess cognitive function including movement-related disorders with high reliability. We studied the relationship between MMSE scores and the ability to take oral medications correctly (ingestion compliance) in 70 elderly inpatients (mean age 71.3+/-7.0 years). Patients with abnormal glucose tolerance as determined by an HbA(1c) level of 5.8% or greater including diabetes showed a trend of lower MMSE scores compared with patients with normal glucose tolerance, and the scores were negatively correlated with HbA1c, age, and systolic blood pressure (P<0.05). Self-management in taking oral medications was very difficult in 4 patients whose MMSE scores were 21 points or less. Thus ingestion supervisions by nurses were required in these patients. Furthermore, 9 of 12 noncompliant patients had MMSE scores ranging from 22 to 26 points. We instructed these patients to take medications in a one-dose package as a useful tool to improve compliance. The MMSE score was 27 or higher in 44 of 54 compliant patients, and 10 patients had scores ranging from 21 to 26. The sensitivity and specificity for noncompliance at an MMSE score cut-off point of 26 were 75.0% and 81.5%, respectively. In conclusion, it is necessary to coordinate ingestion methods matched to each patient according to their abilities to comply with medication schedules. They should be preevaluated with the MMSE to improve ingestion compliance. The MMSE is a recommended test in hospitalized elderly patients for the assessment of the ability to take medications safely.     

Possible relationship between adiponectin and renal tubular injury in diabetic nephropathy

Adiponectin is an adipose-derived protein which has anti-inflammatory and anti-atherogenic properties in addition to insulin-sensitizing effects. To date, the role of adiponectin in the pathogenesis of diabetic nephropathy remains unclear. The aim of the present study was to explore the relationship between adiponectin and renal tubular injury in diabetic nephropathy. We determined serum and urinary adiponectin levels in type 2 diabetic patients with normoalbuminuria (n = 19), microalbuminuria (n = 18), and overt diabetic nephropathy (n = 16), and then analyzed the correlations between serum and urinary adiponectin, urinary N-acetylglucosaminidase (NAG) as a clinical marker of renal tubular injury, urinary monocyte chemoattractant protein-1 (MCP-1) as an inflammatory marker in renal tubulointerstitium, and clinical markers of renal disease. Notably, serum and urinary adiponectin levels were significantly increased in patients with overt diabetic nephropathy compared to those with normoalbuminuria and microalbuminuria. In univariate linear regression analysis, serum adiponectin levels were positively correlated with serum creatinine (r = 0.648, P<0.0001), urinary albumin (r = 0.583, P<0.0001), urinary NAG (r = 0.406, P<0.01), urinary MCP-1 (r = 0.514, P<0.0001), and urinary adiponectin (r = 0.691, P<0.0001) levels in all diabetic patients. Urinary adiponectin levels were also positively correlated with serum creatinine (r = 0.729, P<0.0001), urinary albumin (r = 0.799, P<0.0001), urinary NAG (r = 0.701, P<0.0001), and urinary MCP-1 (r = 0.801, P<0.0001) levels in all diabetic patients. Multiple linear regression analysis showed that serum creatinine and urinary adiponectin levels were independently associated with serum adiponectin levels (r(2) = 0.522), and that serum creatinine, urinary NAG, urinary MCP-1, and serum adiponectin levels were independent determinants of urinary adiponectin levels (r(2) = 0.851). These results collectively indicate that renal insufficiency and tubular injury possibly play a contributory role in increases in serum and urinary adiponectin levels in overt diabetic nephropathy. We presume that an increase in circulating adiponectin in overt diabetic nephropathy might be a physiological response to mitigate renal tubular injury and to prevent the further progression of diabetic nephropathy through its anti-inflammatory and anti-atherogenic effects.     

Aggressive antihypertensive treatment and serum lipid lowering therapy are necessary to prevent deterioration of the renal function even in elderly type 2 diabetic patients with persistent albuminuria

BACKGROUND: In elderly patients with diabetic nephropathy, clinical information aiming at preventing a decline of the renal function is limited. OBJECTIVE: To clarify factors that could be related to changes in renal function in elderly diabetic patients with persistent albuminuria. METHODS: We studied the relationships between the declining rate of creatinine clearance (Ccr) and other clinical features - glycemic control, blood pressure (BP), the degree of albuminuria, and other confounding factors - retrospectively in elderly diabetic patients with persistent albuminuria. RESULTS: We analyzed 17 elderly diabetic patients with persistent albuminuria (4 men and 13 women, mean age 68.0 +/- 5.7 years at the beginning of the observation period) for 3.7 +/- 1.5 (SD) years. We found that the last value of the urinary albumin excretion (UAE) of the observation period and the individual mean values of systolic BP, mean BP, and serum cholesterol closely correlated with the rate of decline in Ccr estimated by Cockcroft's formula. The last UAE closely correlated with the individual mean values of systolic BP and mean BP. For patients such as the present subjects, adequate retardation of decline in renal function requires that the estimated goal of antihypertensive treatment should be a BP below 130/70 mm Hg. The level of glycemic control and the use of angiotensin-converting enzyme inhibitors did not correlate with the rate of decline in Ccr. CONCLUSION: Aggressive antihypertensive treatment and serum lipid lowering therapy may be necessary to prevent deterioration of the renal function in elderly type 2 diabetic patients with persistent albuminuria.     

Methods for preparation and functional analysis of islet beta-cells

Regulation of blood glucose is a fundamental homeostasis in the body. Insulin is released from pancreatic beta-cells in response to changes in blood glucose, the defect of which leads to impaired insulin secretion and diabetes mellitus. Pancreatic beta-cells that release insulin occupy approximately 70% of the islet cells, while alpha, delta, PP-cells are also present in islets. Therefore, analysis of beta-cells is a direct approach for studying mechanisms of insulin secretion. Insulin secretion is regulated by cytosolic Ca(2+) in beta-cells, and its concentration and localization can be measured in real time by fluorescence imaging using indicators. Glucose metabolism is assessed by measurements of NAD(P)H by its autofluorescence. Furthermore, beta-cells are equipped with ion channels, which transduce glucose-evoked metabolic signals to electric signals. Electrophysiological analysis by patch clamp techniques detects the activity of various ion channels and membrane potential. Several beta-cell lines including HIT, MIN6, INS1, RIN, and beta TC are used; however, they do not necessarily retain normal responsiveness to glucose. Therefore, analysis of physiologic functions of beta-cells requires the use of acutely isolated or primary cultured beta-cells from normal animals. The methods for preparation of islets and single beta-cells using collagenase can be applied to a variety of animals of small to large sizes, which can produce islets and beta-cells with physiological responsiveness to glucose. These primary cultured beta-cells can be used for elucidating signal transduction mechanisms, and evaluating effects of drugs, providing excellent tools for physiological, pharmacological, and disease-oriented studies.     

Increased urinary excretion of N-acetylglucosaminidase in subjects with impaired glucose tolerance

N-acetylglucosaminidase (NAG) is a lysosomal enzyme produced by renal proximal tubular cells and has been widely used as a marker, which indicates a degree of renal tubular damage. An increase in urinary NAG excretion is though to result from the renal tubular damage. The aim of this study was to evaluate whether even mild hyperglycemia causes an increase in urinary excretion of NAG, which is a renal tubular protein. We examined urinary NAG excretion in overnight urine in 22 Japanese men with impaired glucose tolerance (IGT) for more than two years (IGT group) and 41 healthy control subjects matched in age, gender, BMI and blood pressure (control group). Urinary NAG excretion levels of IGT group and control group were 2.89 (1.23-7.97) and 2.22 (0.60-4.93) U/g creatinine, median (range), respectively. The IGT group showed significantly higher urinary excretion of NAG compared to the control group (p < 0.01). Several studies have indicated that plasma proteins filtered through the glomerular capillary may have intrinsic renal toxicity. Recently, we have reported that urinary excretion of plasma proteins (ceruloplasmin, IgG4 and IgG) with molecular radii of approximately 45-55 A is increased in subjects with IGT compared to healthy control subjects with normal glucose tolerance. Considering the present result together with our recent finding, we suggest that increased urinary excretion of NAG in the mildly hyperglycemic subjects may be due to the adverse effects of the plasma proteins highly filtered through the glomerular capillary on tubular cells.     

High-resolution electron microscopy of the crystallites of fossil enamels obtained from various geological ages

To elucidate the stability of the central dark line (CDL) in biologically induced hydroxyapatite crystals, we examined the diagenetic changes on the microstructures of the crystallites during the course of fossilization. Using transmission electron microscopy, we investigated the enamel crystallites of fossil animals of various geological ages ranging from Pleistocene to Cretaceous. Electron micrographs indicated that the microstructures and lattice images of each crystallite in fossil enamels were well-preserved regardless of the thickness of the enamel layer, and the presence of CDLs in fossil enamel crystals was also confirmed. The results indicated that the microstructure of hydroxyapatite crystals containing lattice images of CDLs appear stable during long geological periods. In addition, we conclude that the existence of lattice images in apatite with CDLs may be an indicator for the assessment of the evolution of dental enamel from fossil remains.     

Thalamic terminal morphology and distribution of single corticothalamic axons originating from layers 5 and 6 of the cat motor cortex

We investigated the axonal morphology of single corticothalamic (CT) neurons of the motor cortex (Mx) in the cat thalamus, using a neuronal tracer, biotinylated dextran amine (BDA). After localized injection of BDA into the Mx, labeled CT axons were found ipsilaterally in the thalamic reticular nucleus (TRN), the ventroanterior-ventrolateral complex (VA-VL), the central lateral nucleus (CL), the central medial nucleus, and the centromedian nucleus, but with the primary focus in the VA-VL. The terminals in the VA-VL formed a large laminar cluster, which extended approximately in parallel with the internal medullary lamina. The laminar organization mirrored morphologic features of single CT axons. We reconstructed the trajectories of 25 single CT axons that arose from layer V (16 axons) or layer VI (9 axons) and terminated in the VA-VL. Terminals of single CT axons that originated from both layer V and layer VI were confined within a laminar structure about 700 microm thick, suggesting the existence of laminar input organization in the VA-VL. Otherwise, the two groups of the CT axons showed contrasting features. All of the CT axons derived from layer VI gave rise to a few short collaterals to the TRN and then formed extensive arborization with numerous small, drumstick-like terminals in the VA-VL. On the other hand, the CT axons arising from layer V gave rise to collaterals whose main axons descended into the cerebral peduncle. Each collateral projected to the VA-VL or CL without projection to the TRN and formed a few small clusters of giant terminals. The two groups of CT neurons in the same cortical column had convergent rather than segregated termination in the VA-VL. However, the terminals of layer VI CT neurons were distributed diffusely and widely in the VA-VL, whereas the terminals of layer V CT neurons were much more focused and surrounded by the terminals of the former group. These contrasting features of the two types of CT projections appear to represent their different functional roles in the generation of motor commands and control of movements in the Mx.