Kinetic mechanism of Na+, K+, Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions

Summary Ouabain-insensitive, furosemide-sensitive Rb⁺ influx (J _Rb) into HeLa cells was examined as functions of the extracellular Rb⁺, Na⁺ and Cl^– concentrations. Rate equations and kinetic parameters, including the apparent maximumJ _Rb, the apparent values ofK _m for the three ions and the apparentK _i for K⁺, were derived. Results suggested that one unit molecule of this transport system has one Na⁺, one K⁺ and two Cl^– sites with different affinities, one of the Cl^– sites related with binding of Na⁺, and the other with binding of K⁺(Rb⁺). A 11 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of²²Na⁺ and Rb⁺, and a 12 stoichiometry between those of Rb⁺ and³⁶Cl^–. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl^– inhibited ouabain-insensitive Rb⁺ influx, whereas sulfamate and probably also gluconate did not inhibitJ _Rb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na⁺ and one Cl^– bind concurrently to their sites and then one K⁺ (Rb⁺) and another Cl^– bind concurrently. 2) After completion of ion bindings Na⁺, K⁺(Rb^–) and Cl^– in a ratio of 112 show synchronous transmembrane movements.     

Cell surface proteoglycans as a negative modulator in concanavalin a-mediated agglutination of hepatoma cells

Proteoglycan (heparan sulfate-protein conjugate) was solubilized with 8 M urea from rat liver plasma membranes after enzymic (RNAase, neuraminidase) treatments and extensively purified by chromatography and gel filtration. The final products gave an average ratio of hexuronate to protein (weight) of approx. 1.5, contained hexosamine equimolar to hexuronate and were sensitive to β-elimination (the molecular weight being reduced from 20 · 10⁴ to 3 · 10⁴ (gel filtration)).The proteoglycan fraction, when added to trypsinized and untrypsinized ascites hepatoma (AH-130F(N)) cells, inhibited the concanavalin A-mediated agglutination of the cells. However, the alkali-treated proteoglycan (β-elimination) or acid mucopolysaccharide fraction prepared from liver plasma membranes by papain digestion were less effective, and a reference preparation of heparan sulfate was almost ineffective. It was confirmed that significant amounts of proteoglycan labelled with ³⁵SO₄^2? were firmly bound to or taken up by the trypsinized ascites hepatoma cells.These results together with the sensitization of lectin-mediated agglutination by mild protease treatment of cells suggest that cell surface proteoglycans may act as a negative modulator in the lectin-mediated agglutination of cells.     

Evaluation of Fucosylated Haptoglobin and Mac-2 Binding Protein as Serum Biomarkers to Estimate Liver Fibrosis in Patients with Chronic Hepatitis C

Fucosylated haptoglobin (Fuc-Hpt) and Mac-2 binding protein (Mac-2 bp) are identified as cancer biomarkers, based on the results from a glyco-proteomic analysis. Recently, we reported that these glyco-biomarkers were associated with liver fibrosis and/or ballooning hepatocytes in patients with nonalcoholic fatty liver disease (NAFLD). We evaluated the ability of these glycoproteins to estimate liver fibrosis in 317 patients with chronic hepatitis C. We measured the serum Fuc-Hpt and Mac-2 bp levels using a lectin-antibody ELISA and ELISA, respectively. The serum levels of both Fuc-Hpt and Mac-2 bp increased with the progression of liver fibrosis. The multivariate analysis revealed that Mac-2 bp was an independent factor associated with moderate liver fibrosis (F ≥ 2). In contrast, Fuc-Hpt was an independent factor associated with advanced liver fibrosis (F ≥ 3). In terms of evaluating liver fibrosis, the serum levels of these glycomarkers were correlated with well-known liver fibrosis indexes, such as the aspartate aminotransferase to platelet ratio index (APRI) and Fibrosis-4 (FIB4) index. An assay that combined the APRI or FIB4 index and the Fuc-Hpt or Mac-2 bp levels increased the AUC value for diagnosing hepatic fibrosis. Interestingly, the cumulative incidence of hepatocellular carcinoma (HCC) was significantly higher in the patients with elevated serum levels of Fuc-Hpt and Mac-2 bp. In conclusion, both Fuc-Hpt and Mac-2 bp could be useful glyco-biomarkers of liver fibrosis and predictors of HCC in patients with chronic hepatitis C.     

Chronic kidney disease after 5/6 nephrectomy disturbs the intestinal microbiota and alters intestinal motility

Organ–organ crosstalk is involved in homeostasis. Gastrointestinal symptoms are common in patients with renal failure. The aim of this study was to elucidate the relationship between gastrointestinal motility and gastrointestinal symptoms in chronic kidney disease. We performed studies in C57BL/6 mice with chronic kidney disease after 5/6 nephrectomy. Gastrointestinal motility was evaluated by assessing the ex vivo responses of ileum and distal colon strips to electrical field stimulation. Feces were collected from mice, and the composition of the gut microbiota was analyzed using 16S ribosomal RNA sequencing. Mice with chronic kidney disease after 5/6 nephrectomy showed a decreased amount of stool, and this constipation was correlated with a suppressed contraction response in ileum motility and decreased relaxation response in distal colon motility. Spermine, one of the uremic toxins, inhibited the contraction response in ileum motility, but four types of uremic toxins showed no effect on the relaxation response in distal colon motility. The 5/6 nephrectomy procedure disturbed the balance of the gut microbiota in the mice. The motility dysregulation and constipation were resolved by antibiotic treatments. The expression levels of interleukin 6, tumor necrosis factor-α, and iNOS in 5/6 nephrectomy mice were increased in the distal colon but not in the ileum. In addition, macrophage infiltration in 5/6 nephrectomy mice was increased in the distal colon but not in the ileum. We found that 5/6 nephrectomy altered gastrointestinal motility and caused constipation by changing the gut microbiota and causing colonic inflammation. These findings indicate that renal failure was remarkably associated with gastrointestinal dysregulation.     

Two species of <Emphasis Type="Italic">Leptographium</Emphasis> isolated from blue-stained sapwood of <Emphasis Type="Italic">Pinus khasya</Emphasis> and bark beetles in Thailand

Two species of Leptographium were isolated from blue-stained sapwood of Pinus khasya and bark beetle galleries in pine trees near Chiang Mai, Thailand. Based on morphological observations, these two species were identified as L. pini-densiflorae and L. yunnanense. This is the first record of these fungi in Thailand. Leptographium yunnanense appeared to be associated with Polygraphus major. This is contribution No. 206, Laboratory of Plant Parasitic Mycology, Graduate School of Life & Environmental Sciences, University of Tsukuba     

Evolutionary developmental biology and genomics

Reciprocal questions often frame studies of the evolution of developmental mechanisms. How can species share similar developmental genetic toolkits but still generate diverse life forms? Conversely, how can similar forms develop from different toolkits? Genomics bridges the gap between evolutionary and developmental biology, and can help answer these evo-devo questions in several ways. First, it informs us about historical relationships, thus orienting the direction of evolutionary diversification. Second, genomics lists all toolkit components, thereby revealing contraction and expansion of the genome and suggesting mechanisms for evolution of both developmental functions and genome architecture. Finally, comparative genomics helps us to identify conserved non-coding elements and their relationship to genome architecture and development.     

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells. In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-l-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.