The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic β cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucose absorption in vivo was measured by single-pass perfusion method. Incorporation of [¹⁴C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [¹⁴C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin-mediated pathway rather than through a GLP-1-mediated pathway.     

H1.X with different properties from other linker histones is required for mitotic progression

We report here the characterization of H1.X, a human histone H1 subtype. We demonstrate that H1.X accumulates in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. In addition, the results of fluorescence recovery after photobleaching indicate that the exchange of H1.X on and off chromatin is faster than that of the other H1 subtypes. Furthermore, RNA interference experiments reveal that H1.X is required for chromosome alignment and segregation. Our results suggest that H1.X has important functions in mitotic progression, which are different from those of the other H1 subtypes.     

The antimicrobial peptide, psacotheasin induces reactive oxygen species and triggers apoptosis in Candida albicans

Previously, the antimicrobial effects and membrane-active action of psacotheasin in Candida albicans were investigated. In this study, we have further found that a series of characteristic cellular changes of apoptosis in C. albicans can be induced by the accumulation of intracellular reactive oxygen species, specifically hydroxyl radicals, the well-known important regulators of apoptosis. Cells treated with psacotheasin showed diagnostic markers in yeast apoptosis at early stages: phosphatidylserine externalization from the inner to the outer membrane surface, visualized by Annexin V-staining; mitochondrial membrane depolarization, observed by DiOC₆(3) staining; and increase of metacaspase activity, measured using the CaspACE FITC-VAD-FMK. Moreover, DNA fragmentation and condensation also revealed apoptotic phenomena at late stages through the TUNEL assay staining and DAPI staining, respectively. Taken together, our findings suggest that psacotheasin possess an antifungal property in C. albicans via apoptosis as another mode of action.     

Structural requirements of chromokinesin Kif4A for its proper function in mitosis

Human Kif4A is a member of the Kinesin-4 family of kinesins. Kif4A is thought to be a bona fide chromokinesin because it possesses a motor domain and associates with condensed chromosomes during mitosis. Genetic deletion of Kif4A promotes tumorigenic phenotypes in mouse embryonic cells. Kif4A is critical for mitotic regulation including chromosome condensation, spindle organization and cytokinesis. However, the precise chromatin-binding domain of Kif4A has not been characterized. Herein, we report the identification of two conserved motifs critical for chromatin-binding: the first leucine Zip motif (Zip1) of a leucine Zip/Basic/leucine Zip region (ZBZ) previously thought to be a nuclear localization signal (NLS), and a cysteine-rich (CR) motif within the C-terminal region of Kif4A. Furthermore, by depleting endogenous Kif4A via RNAi and concurrently expressing RNAi-resistant Kif4A versions, we observed that wild type Kif4A, but not the mutants deficient in DNA-binding (Zip1 or CR deleted) or ATPase activity (K94A point mutant), was able to rescue the RNAi-elicited abnormal mitotic profile. Taken together, our results show that both the Zip1 and CR motifs are important for Kif4A chromatin-binding and its mitotic function.     

Control of growth cone motility and neurite outgrowth by SPIN90

SPIN90 is an F-actin binding protein thought to play important roles in regulating cytoskeletal dynamics. It is known that SPIN90 is expressed during the early stages of neuronal development, but details of its localization and function in growth cones have not been fully investigated. Our immunocytochemical data show that SPIN90 is enriched throughout growth cones and neuronal shafts in young hippocampal neurons. We also found that its localization correlates with and depends upon the presence of F-actin. Detailed observation of primary cultures of hippocampal neurons revealed that SPIN90 knockout reduces both growth cone areas and in the numbers of filopodia, as compared to wild-type neurons. In addition, total neurite length, the combined lengths of the longest (axonal) and shorter (dendritic) neurites, was smaller in SPIN90 knockout neurons than wild-type neurons. Finally, Cdc42 activity was down-regulated in SPIN90 knockout neurons. Taken together, our findings suggest that SPIN90 plays critical roles in controlling growth cone dynamics and neurite outgrowth.     

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.     

Induction of yeast apoptosis by an antimicrobial peptide, Papiliocin

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC₆(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation.     

Both plant and animal LEA proteins act as kinetic stabilisers of polyglutamine-dependent protein aggregation

LEA (late embryogenesis abundant) proteins are intrinsically disordered proteins that contribute to stress tolerance in plants and invertebrates. Here we show that, when both plant and animal LEA proteins are co-expressed in mammalian cells with self-aggregating polyglutamine (polyQ) proteins, they reduce aggregation in a time-dependent fashion, showing more protection at early time points. A similar effect was also observed in vitro, where recombinant LEA proteins were able to slow the rate of polyQ aggregation, but not abolish it altogether. Thus, LEA proteins act as kinetic stabilisers of aggregating proteins, a novel function in protein homeostasis consistent with a proposed role as molecular shields.     

Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S)

A human isoform of the vesicle-associated membrane protein-associated proteins (VAPs), VAPB, causes amyotrophic lateral sclerosis eight due to the missense mutation of Pro-56, whereas human VAPA and the yeast VAP Scs2p proteins are not significantly affected by similar mutations. We have found that VAPA and Scs2p have three prolines present in a conserved region however VAPB has only two prolines in this region. Consequently, this mutation in VAPB (VAPB(P56S)) leaves a single proline in this region whereas other VAPs can retain two proline residues even if the proline equivalent to the Pro-56 is substituted. When Scs2p and VAPA were mutated to be equivalent to VAPB(P56S) in terms of the distribution of proline residues in this region, Scs2p became inactive and aggregated, and VAPA localize to membranous aggregates indistinguishable from those induced by VAPB(P56S). This suggests that the appropriate distribution of three conserved prolines, not the existence of a particular proline, confers VAPA and Scs2p resistance to the Pro-56 mutation and, therefore, is critical for VAP activities.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.     

Age-dependent guanine oxidation in DNA of different brain regions of Wistar rats and prematurely aging OXYS rats

Background

Oxidative damage to the cell, including the formation of 8-oxoG, has been regarded as a significant factor in carcinogenesis and aging. An inbred prematurely aging rat strain (OXYS) is characterized by high sensitivity to oxidative stress, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases including learning and memory deterioration.

Methods

We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) and 8-oxoguanine DNA glycosylase (OGG1) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative amounts and distribution of 8-oxoG and OGG1 in various cells of different brain regions from OXYS and control Wistar rats.

Results

It was shown that 8-oxoG increased with age in mature neurons, nestin- and glial fibrillary acidic protein (GFAP)-positive cells of hippocampus and frontal cortex in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. The relative content of 8-oxoG and OGG1 in nestin- and GFAP-positive cells was higher than in mature neurons in both Wistar and OXYS rats. However, there was no significant interstrain difference in the content of OGG1 for all types of cells and brain regions analyzed, and no difference in the relative content of 8-oxoG between different brain regions.

Conclusions

Oxidation of guanine may play an important role in the development of age-associated decrease in memory and learning capability of OXYS rats.

General significance

The findings are important for validation of the OXYS rat strain as a model of mammalian aging.     

Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood.     

Structure and Function of Palladin's Actin Binding Domain

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.     

The Tudor Domain of the PHD Finger Protein 1 Is a Dual Reader of Lysine Trimethylation at Lysine 36 of Histone H3 and Lysine 27 of Histone Variant H3t

PHF1 associates with the Polycomb repressive complex 2 and it was demonstrated to stimulate its H3K27-trimethylation activity. We studied the interaction of the PHF1 Tudor domain with modified histone peptides and found that it recognizes H3K36me3 and H3tK27me3 (on the histone variant H3t) and that it uses the same trimethyllysine binding pocket for the interaction with both peptides. Since both peptide sequences are very different, this result indicates that reading domains can have dual specificities. Sub-nuclear localization studies of full-length PHF1 in human HEK293 cells revealed that it co-localizes with K27me3, but not with K36me3, and that this co-localization depends on the trimethyllysine binding pocket indicating that K27me3 is an in vivo target for the PHF1 Tudor domain. Our data suggest that PHF1 binds to H3tK27me3 in human chromatin, and H3t has a more general role in Polycomb regulation.     

New function of the proline rich domain in dynamin-2 to negatively regulate its interaction with microtubules in mammalian cells

Microtubule reorganization is necessary for many cellular functions such as cell migration, cell polarity and cell division. Dynamin was originally identified as a microtubule-binding protein. Previous limited digestion experiment revealed that C-terminal 100-amino acids proline rich domain (PRD) of dynamin is responsible for microtubule binding in vitro. However, as obvious localization of dynamin along microtubules is only observed at the spindle midzone during mitosis but not in interphase cells, it remains unclear how dynamin interacts with microtubules in vivo. Here, we report that GFP-dynamin-2-(1-786), a truncated mutant lacking a C-terminal portion of the PRD, localized along microtubules in interphase HeLa cells. GFP-dynamin-2-wild type (WT) and GFP-dynamin-2-(1-745), a construct that was further truncated to remove the entire PRD, localized in discrete punctate structures but not along microtubules. These data suggest that the N-terminal (residues 746-786) but not the entire PRD is necessary for the interaction of dynamin-2 with microtubules in the cell and that the C-terminus of PRD (787-870) negatively regulate this interaction. Microtubules in cells expressing GFP-dynamin-2-(1-786) were stabilized against exposure to cold. These results provide a first evidence for a regulated interaction of dynamin-2 with microtubules in cultured mammalian cells.