Interactions between Age-Related Type 2 Diabetes and the Small Intestine.

The number of patients with type 2 diabetes is increasing worldwide. The mechanisms leading to type 2 diabetes and its complications is being researched; however, the pathological mechanisms of diabetes in the small intestine remain unclear. Therefore, we examined these pathological mechanisms in the small intestine using a mouse model of type 2 diabetes (KK-Ay/TaJcl) aged 10 and 50 weeks. The results showed that diabetes worsened with age in the mice with type 2 diabetes. In these mice, advanced glycation end products (AGEs) in the small intestine and mast cell expression increased, whereas diamine oxidase (DAO) decreased; increased tumor necrosis factor (TNF)-α and histamine levels in the plasma and small intestine were also detected. Additionally, the expression of zonula occludens (ZO)-1 and Claudin1 and cell adhesion molecules in the small intestine reduced. These results exacerbated with age. These findings indicate that type 2 diabetes causes AGE/mast cell/histamine and TNF-α signal transmission in the small intestine and decreases small intestinal wall cell adhesion molecules cause TNF-α and histamine to flow into the body, worsening the diabetic condition. In addition, this sequence of events is suggested to be strengthened in aged mice with type 2 diabetes, thus exacerbating the disease. These findings of this study may facilitate the elucidation of the pathological mechanisms of type 2 diabetes and its associated complications.

Association between Weight Gain and Sex-Related Differences through 5-Fluorouracil Administration.

Research on sex differences has increased across various fields, including cancer and its treatment domains. Reports have indicated sex differences in cancer incidence, survival rates, and the efficacy of anticancer drugs. However, such reports are limited, and in-depth assessments of the underlying mechanisms are still in progress. Although various chemotherapeutic regimens are applicable for breast cancer treatment, reports have surfaced regarding weight gain in female patients undergoing fluorouracil, epirubicin, cyclophosphamide (FEC) or cyclophosphamide, methotrexate, fluorouracil (CMF) therapy. We hypothesized the potential of 5-fluorouracil (5-FU) in weight gain and sex-related differences. To address this, we conducted experiments in mice to confirm weight gain and sex differences following 5-FU administration, and elucidate the underlying mechanisms. Our findings revealed weight gain and increased food intake in female mice following 5-FU administration. Additionally, female mice receiving 5-FU exhibited increased norepinephrine and α1- and α2-adrenergic receptor expression, reduced estradiol levels, and increased ghrelin levels. These results indicate 5-FU administration-induced sex differences in weight gain and implicate increased food intake because of increased norepinephrine and α1- and α2-adrenergic receptor expression, reduced estradiol levels, and a subsequent increase in ghrelin levels, which contribute to weight gain in female patients undergoing CMF therapy.

ACTH/cAMP-Mediated Skin Pigmentation Caused by 5-Fluorouracil Administration.

Anticancer drugs exhibit many side effects, including skin pigmentation, which often lowers patient QOL. However, the mechanism of pigmentation caused by anticancer drugs remains unknown. The purpose of this study was to elucidate the mechanism of anticancer drug-induced skin pigmentation using 5-fluorouracil (5-FU), a widely used anticancer drug. Specific pathogen-free, 9-week-old Hos:HRM-2 male mice were intraperitoneally administered 5-FU daily for 8 weeks. Skin pigmentation was observed at the end of the study. Mice treated with 5-FU were also administered inhibitors of cAMP, α-melanocyte-stimulating hormone (α-MSH), and adrenocorticotropic hormone (ACTH) for analysis. Administration of oxidative stress, nuclear factor-kappa B (NF-κB), cAMP, and ACTH inhibitors reduced pigmentation in 5-FU-treated mice. These results indicate that the oxidative stress/NF-κB/ACTH/cAMP/tyrosinase pathway plays an important role in pigmentation in 5-FU-treated mice.

Dark-Field Microscopic Detection of Bacteria using Bacteriophage-Immobilized SiO2@AuNP Core-Shell Nanoparticles.

To replace molecular biological and immunological methods, biosensors have recently been developed for the rapid and sensitive detection of bacteria. Among a wide variety of biological materials, bacteriophages have received increasing attention as promising alternatives to antibodies in biosensor applications. Thus, we herein present a rapid and highly selective detection method for pathogenic bacteria, which combines dark-field light scattering imaging with a plasmonic biosensor system. The plasmonic biosensor system employs bacteriophages as the biorecognition element and the aggregation-induced light scattering signal of gold nanoparticle-assembled silica nanospheres as a signal transducer. Using Staphylococcus aureus strain SA27 as a model analyte, we demonstrated that the plasmonic biosensor system detects S. aureus in the presence of excess Escherichia coli in a highly selective manner. After the sample and the S. aureus phage S13'-conjugated plasmon scattering probe were mixed, S. aureus detection was completed within 15-20 min with a detection limit of 8 × 104 colony forming units per milliliter.

Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules.

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

Dementia Is Induced via the AGEs/Iba1/iNOS Pathway in Aged KK-Ay/Tajcl Mice.

The onset and exacerbation of dementia have been observed in elderly patients with type 2 diabetes. However, the underlying mechanism remains unclear. In this study, we investigated the effects of aging on the cognitive function in a mouse model of type 2 diabetes. Pathogen-free KK-Ay/TaJcl mice were used in this study. The cognitive abilities and memory declined in the mice and worsened in the 50-week-olds. The levels of advanced glycation end products (AGEs), receptor for AGE (RAGE), and Iba1 in the hippocampus were increased in the mice compared to those in the control mice. Hippocampal levels of CC-chemokine receptor 7 and inducible nitric oxide synthase, which are from M1-type macrophages that shift from microglia, were higher in KK-Ay/TaJcl mice than in control mice. Tumor necrosis factor (TNF)-α and nitric oxide (NO) levels secreted by M1-type macrophages were similarly elevated in the mice and were even higher at the age of 50 weeks. NO levels were markedly elevated in the 50-week-old mice. In contrast, differentiation of CD163 and arginase-1 did not change in both mouse types. Memory and learning declined with age in diabetic mice, and the AGEs/RAGE/M1-type macrophage/NO and TNF-α pathways played an important role in exacerbating memory and learning in those mice.

Changes in the AGE/Macrophage/TNF-α Pathway Affect Skin Dryness during KK-Ay/Tajcl Mice Aging.

Skin dryness associated with type 2 diabetes worsens with age; however, the underlying mechanisms remain unclear. Herein, we investigated the effects of aging on skin dryness using a type 2 diabetes mice model. Specific pathogen-free KK-Ay/TaJcl mice of different ages (10, 27, 40, and 50 weeks) were used in this study. The results confirmed that skin dryness worsens with age. Furthermore, increased levels of advanced glycation end products (AGE), prostaglandin E2 (PGE2), and tumor necrosis factor (TNF)-α, along with an increased expression of the major AGE receptor (RAGE), an increased macrophage number, and decreased collagen expression were observed in the skin of aged KK-Ay/TaJcl mice. In conclusion, dry skin conditions worsen with age in diabetic mice, and the AGE/RAGE/PGE2 and TNF-α pathways play an important role in exacerbating skin dryness during aging in these mice.

Development of an Automatic Chronological Record Creation System Using Voice AI to Facilitate Information Aggregation and Sharing in the Event of a Disaster.

OBJECTIVE: In the event of a disaster, the chain of command and communication of each relevant agency is important. In this study, a chronological record creation system using voice AI (V-CRS) was developed, and an experiment was conducted to determine whether the obtained information could be quickly and easily summarized in chronological order. METHODS: After a lecture by Japanese Disaster Medical Assistant (DMAT) Team members and 8 medical clerks on how to use the developed tool, a comparison experiment was conducted between manual input and V-CRS utilization of the time to compile disaster information. RESULTS: Results proved that V-CRS can collect information gathered at headquarters more quickly than handwriting. It was also suggested that even medical clerks who have never been trained to record information during disasters could record information at the same speed as trained DMAT personnel. CONCLUSION: V-CRS can transcribe audio information even in situations where technical terms and physical units must be recorded, such as radiation disasters. It has been proven that anyone can quickly organize information using this method, to some extent.

Mechanism of Cd-induced inhibition of Na-glucose cotransporter in rabbit proximal tubule cells: roles of luminal pH and membrane-bound carbonic anhydrase.

BACKGROUND/AIMS: We have previously reported that a complex of cadmium-metallothionein (Cd-MT) directly affects the apical Na-glucose cotransporter on the luminal side in proximal tubules, suggesting that Cd-MT is more toxic than CdCl(2) in causing tubulopathy. To find the potential mechanisms, we evaluated the effect of luminal pH alteration and carbonic anhydrase (CA) inhibition on Cd-MT-induced reduction of glucose-dependent transmural voltage in rabbit S2 segments perfused in vitro. METHODS: Before and after the addition of Cd-MT (1 microg Cd/ml) to the lumen, the deflections of transmural voltage upon the elimination of glucose from the perfusate (DeltaVt(glu)) were measured as a parameter of activity of the Na-glucose cotransporter. RESULTS: During perfusion with a control solution of pH 7.4, the DeltaVt(glu) significantly decreased after addition of Cd-MT for 10 min. A reduction in pH to 6.8 significantly shortened the time needed to reduce the DeltaVt(glu) to <5 min, whereas an increase of pH to 7.7 significantly prolonged the time to >20 min. Furthermore, simultaneous addition of acetazolamide with control perfusate prevented the reduction. P-Fluorobenzyl-aminobenzolamide (pFB-ABZ), a membrane-impermeable CA inhibitor, added to the lumen also completely prevented the reduction in DeltaVt(glu). In rabbits with chronic Cd exposure, acetazolamide prevented the glucosuria. CONCLUSION: Cd-MT-induced inhibition of Na-glucose cotransporter activity in the S2 segment strongly depends on luminal pH, and that an increase in pH by inhibition of luminal membrane-bound CA is useful to prevent renal Cd toxicity.

Cloning and identification of a new member of water channel (AQP10) as an aquaglyceroporin.

Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally.

Opening of ligand-gated cation channel families by calpain inhibitors.

The class of Ca2+-permeable cation channels is composed of large families with six transmembrane segments including transient receptor potential, vanilloid receptor (VR), polycystin, epithelial calcium channels and melastatin (MLS). However, most of them are functionally silent and unexpressed in mammalian cells. An investigation of associated proteins made us believe that the blockade of calpain opens the silent channels. Using 1 microM of blockers in whole cellular patch pipette fill we measured currents of Chinese hamster ovary cells transfected by VR-like 1 and 2, polycystin-2, or a MLS-like new member (MLS3S). Significant conductance of every clone with a characteristic rectification by blockers was demonstrated. The permeability of Ca2+ to them is similar to that reported. Western blot suggested that blockers did not affect the assembly of the protein but enabled its cleavage. Therefore, investigation of these families with the blockers may boost our knowledge of electrophysiologic function.

Effects of various culture environments on expression of major outer membrane proteins from Porphyromonas gingivalis.

We examined the effects of various culture environments on major outer membrane proteins from Porphyromonas gingivalis ATCC 33277. Major outer membrane protein patterns on gel electrophoresis showed little difference over the culturable range of osmolarity and pH. With elevated temperature or prolonged culture, the intensities of the gingipain bands decreased; however, bands of RagA, RagB and the putative porins were relatively stable. Similar results were observed with several different culture media. Although the precise functions of RagA, RagB and the putative porins are unknown, these factors may be strongly related to the initiation and progression of adult periodontitis.

Identification of a new member of type I Na/phosphate co-transporter in the rat kidney.

AIM: To clone new phosphate transporters from the kidney. METHODS: EST bank was screened for new members of Na-dependent inorganic phosphate (P(i)) co-transporter. To obtain a full-length clone, a rat kidney cDNA library was screened with the EST clone as a probe. RESULTS: We isolated a cDNA that encodes a type I Na/P(i) co-transporter. Based on the sequence homology and tissue distribution, it will be the rat ortholog of human NPT4 (Solute Carrier family 17, member A3, SLC17A3) and is designated rNPT4. When expressed in Xenopus oocytes, Na/P(i) co-transport activity was demonstrated. In Northern blotting analysis, rNPT4 was selectively expressed in the liver and kidney. In immunohistochemical analysis, rNPT4 was localized at the apical membrane of the proximal tubules (S2, S3) in the rat kidney. The electron microscopic studies confirmed this finding. CONCLUSION: The present study revealed the expression of a new type I Na/P(i) co-transporter at the brush border membrane of the proximal tubules in the kidney.

Primary cell preparation of human renal tubular cells for transcriptome analysis.

We initiated a toxicogenomics project using Affymetrix GeneChip((R)) HG-U133A and HG-U133B arrays harboring 45,000 probe sets representing more than 39,000 transcripts to analyze gene expression in primary cultures of human cells after exposure to chemicals that cause tissue toxicity. In order to assess the quality of the samples studied, we prepared primary human renal cortical cell cultures from surgically resected human kidney and evaluated the origin of the cells and the effects of cryopreservation. We analyzed the primary cultures using GeneChip and compared their expression patterns with those in the Novartis Research Foundation (GNF) Gene Expression Database. The comparison with the GNF database revealed that the gene expression pattern of the cultured cells was compatible with kidney cells, indicating that we had purified human renal cortical cells. Due to the purification procedure, the primary cultured cells could be a mixture of renal components; however, we identified the major population as renal proximal tubule cells by assessing gamma-GTP activity and Glut2 antigen expression. We compared gene expression in the cells before and after cryopreservation. The expression of 567 selected housekeeping genes was unchanged by cryopreservation (Pearson's correlation coefficient r = 0.980; p < 0.0001). The analysis of more than 39,000 transcripts after normalization revealed no significant changes in expression. These results indicate that our method is satisfactory for obtaining adequate primary cell cultures of renal origin and that gene expression was not significantly changed by cryopreservation.

Computer analysis of the significance of the effective osmolality for urea across the inner medullary collecting duct in the operation of a single effect for the counter-current multiplication system.

BACKGROUND: Although urea and water are transported across separate pathways in the apical membrane of the inner medullary collecting duct (IMCD), the existence of a cellular diffusion barrier as an unstirred layer makes it possible to use coefficients of effective osmotic force (sigma*) as equivalent to reflection coefficients. The difference in effective osmolality between urea and NaCl across the IMCD becomes a driving force for water if the compositions of solutes are different between tubular lumen and interstitium. Since reported values for sigma*(urea) are discrepant, we compared the efficiency of a single effect in the counter-current system between an ascending thin limb (ATL) and the IMCD, with the interposition of capillary networks (CNW), between two models with sigma(urea)* = 0.7 (model 1) and sigma(urea)* = 1.0 (model 2). METHODS: The time courses (within 3 s) of solute and the water transport profiles among ATL, CNW, and IMCD were simulated with a computer in the absence of flow in each compartment. RESULTS: In spite of small differences in the profiles of urea and NaCl concentrations between the two models, model 1 displayed a larger volume flux in the IMCD than model 2, resulting in an increase of osmolality in the IMCD and a decrease of osmolality in the ATL. These findings are vital for the operation of the counter-current multiplication system. CONCLUSIONS: The concept of coefficients for effective osmotic force can be applied to the counter-current model between the IMCD and the ATL with the interposition of CNW. The model of sigma(urea)* = 0.7 is more efficient than that of sigma(urea)* = 1.0.

Mechanism of iodide transport in the rabbit cortical collecting duct.

BACKGROUND: Pendrin, an anion exchanger known to participate in iodide transport in the apical membrane of follicular cells of the thyroid gland, has recently been shown to exist in the apical membrane of the beta- and gamma-intercalated (beta/gamma-IC) cells of the cortical collecting duct (CCD). We examined mechanisms of iodide transport in the CCD. METHODS: Rabbit CCD was perfused in vitro, and lumen-to-bath flux coefficients for both (125)I(-) (K(I (lb))) and (36)Cl(-) (K(Cl (lb))) were measured simultaneously. The intracellular pH (pHi) of beta/gamma-IC cells in the perfused CCD was measured by microscopic fluorometory, by loading 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxy methylester (BCECF-AM), a fluorescent marker for pHi. The effects on pHi of the replacement of NaCl with Na cyclamate, NaI, or NaBr in the lumen or bath were observed. RESULTS: K(I (lb)) was comparable to or slightly higher than K(Cl (lb)). Both iodide and chloride in the lumen caused self- and cross-inhibitions to both fluxes. The addition of 5-nitro-2-(-3-phenylpropylamino)-benzoate (NPPB), a Cl(-) channel inhibitor, to the bath significantly reduced K(Cl (lb)), but not K(I (lb)). Replacement of luminal fluid NaCl with Na cyclamate, NaI, or NaBr caused alkalization of pHi, no change in pHi, and slight acidification of pHi, respectively. Replacement of bath NaCl with Na cyclamate, NaI, or NaBr caused alkalization, alkalization, and acidification of pHi, respectively. Luminal NaI prevented the acidification of pHi caused by bath Na cyclamate. CONCLUSIONS: The data are consistent with the model that iodide is transported via the Cl(-)/HCO(3) (-) exchanger in the apical membrane of beta/gamma-IC cells and exits the basolateral membrane via an electroneutral transporter that is distinct from the Cl(-) channel. We could not, however, identify which type of beta/gamma-IC cell was mainly responsible.

Major outer membrane proteins from Porphyromonas gingivalis: strain variation, distribution, and clinical significance in periradicular lesions.

Porphyromonas gingivalis has been implicated in both marginal periodontitis and periapical infection. This study examined the major outer membrane proteins, from P. gingivalis, which related to periradicular lesions. Outer membrane protein profiles of P. gingivalis ATCC 33277 and W83 were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis. Most outer membrane proteins, such as RagA, gingipains, and OmpA-like proteins, were found in both strains in a similar distribution pattern; however, the migration positions of Lys-gingipain and RagB were inverted in SDS-PAGE. Western blot analysis showed that RagA, RagB, and OmpA-like proteins were found in all of the P. gingivalis strains tested. The antiserum of W83 against RagB reacted poorly to some strains, such as ATCC 33277. When strains phylogenetically related to P. gingivalis were examined, RagA and OmpA homologs were immunologically detected in several strains. However, none of the RagB homologs were detected in any strain analyzed, suggesting that RagB is unique to P. gingivalis. To examine immunoreactive antigens in P. gingivalis, sera from patients with periradicular lesions were used. More than half of the sera showed strong reactions to P. gingivalis cell components, especially RagB. Our results indicate that a major outer membrane protein, RagB, is a possible virulence factor in periradicular lesions.