Transfection of Corynebacterium glutamicum with Temperate Phage ϕ CG1

Versatile plasmid vectors useful for gene cloning in Brevibacterium lactofermentum, a glutamic acid-producing bacterium, have been constructed. The trimethoprim (Tp)-resistant dihydrofolate reductase gene derived from chromosomal DNA of the Tp-resistant mutant of B. lactofermentum was introduced into pAM330, a cryptic plasmid in B. lactofermentum. The constructed cloning vector pAJ228 (7.6kb) exists in 10 to 20 copies in cells of B. lactofermentum and donated Tp-resistance, which is a useful selective marker of transformants. pAJ228 was further improved to a versatile plasmid vector pAJ224 having some profitable characteristics such as smaller size (3.7 kb), higher copy number (60 ~ 80 copies), and additional useful cloning sites (Bam HI, Pst I and Sal I) equipped with two different promoters arranged at both orientations for the expression of passenger DNA without promoter. These plasmids were stably retained in B. lactofermentum even in the absence of Tp over many generations. Thus, they have been found very powerful vectors for gene cloning in Brevibacterium and the related bacteria.     

Elucidation of proliferative capability of mononuclear tetraploid cells,emerging spontaneously from diploid cells,using image cytometry and fluorescence in situ hybridization

Objectives

Proliferation of tetraploid cells (TCs) emerging from diploid cells is considered to be a critical event toward tumourigenesis, or cancer progression. Recently, several studies have reported that binuclear TCs emerging from normal cells are capable of mitosis, however, it has not been confirmed directly whether mononuclear TCs emerging from normal cells could proliferate, even cancer cells. The aim of this study is to detect mononuclear TCs in vitro, spontaneously emerging from diploid cells and to elucidate their proliferative capability directly. For this purpose, we have developed a novel method.

Materials and methods

In this study, two completely disomic cell lines were used, TIG‐7, a fibroblast cell line and CAL‐51, a breast cancer cell line. Cells were cultured on microscope slides and their DNA content was determined using an image cytometer. On the same slides, chromosome numbers were scored using centromere fluorescence in situ hybridization (FISH). For evaluating proliferative capability of TCs, bromodeoxyuridine (BrdUrd) incorporation and colony‐forming ability were examined.

Results

Using our method, spontaneous emergence of mononuclear TCs was detected in both TIG‐7 and CAL‐51. Colonies of TIG‐7 TCs were not observed, but were observed of CAL‐51 TCs.

Conclusions

Our method enables detection of mononuclear TCs and elucidation of their proliferative capability, directly; this evidence reveals that mononuclear TIG‐7 TCs do not proliferate but that mononuclear CAL‐51 TCs are able to.

     

MK-801 alters diaphragmatic activities in unanesthetized rats differently between normoxia and hypoxia

This study was designed to examine how systemic administration of an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, altered respiratory timing in unanesthetized rats under normoxia and hypoxia. To detect fine changes in inspiratory time (TI) and expiratory time (TE), and cycle duration (TTOT), we prepared a diaphragmatic electromyogram (EMGdia). Diaphragm electrodes and arterial and venous catheters were inserted into Wistar rats (n = 8) under pentobarbital anesthesia. The next day, EMGdia was recorded before and after intravenous administration of MK-801 (3 mg/kg) under normoxia and hypoxia (12% O2) without anesthesia, and the respiratory timing (TI, TE, TTOT), respiratory frequency (fR), and amplitude of the integrated EMGdia were measured. Arterial blood gases (ABGs), mean arterial pressure (MAP), and heart rate (fH) were also measured with the EMGdia. Under normoxia, MK-801 increased fR owing to a significant decrease in TE, and elevated both MAP and fH. Under hypoxia, MK-801 suppressed an increase in fR owing to a significant increase in TI, and did not accelerate fH. In both gaseous conditions, on ABGs, MK-801 did not alter partial pressure of O2 (PaO2) or CO2 (PaCO2), and slightly decreased pH (but not less than 7.4). MK-801 significantly decreased hypoxic response (%change from normoxia) in fR, and increased that in EMGdia amplitude, and did not alter a total ventilatory index (fRxEMGdia amplitude). The results suggest that an NMDA receptor-mediated mechanism partially determines fR through significant alterations in respiratory timing, particularly in which the hypoxic ventilatory response was obtained in unanesthetized rats.     

Very-KIND, a KIND domain containing RasGEF, controls dendrite growth by linking Ras small GTPases and MAP2

The regulation of cytoskeletal components in the dendritic shaft core is critical for dendrite elongation and branching. Here, we report that a brain-specific Ras guanine nucleotide exchange factor (RasGEF) carrying two kinase non-catalytic C-lobe domains (KINDs), very-KIND (v-KIND), regulates microtubule-associated protein 2 (MAP2). v-KIND is expressed in developing mouse brain, predominantly in the cerebellar granule cells. v-KIND not only activates Ras small GTPases via the C-terminal RasGEF domain, but also specifically binds to MAP2 via the second KIND domain (KIND2), leading to threonine phosphorylation of MAP2. v-KIND overexpression suppresses dendritic extension and branching of hippocampal neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression promotes dendrite growth. These findings suggest that v-KIND mediates a signaling pathway that links Ras and MAP2 to control dendrite growth.     

An Osteoprotegerin Gene Polymorphism Is Associated with an Increased Risk of Hip Fracture in Japanese Patients with Rheumatoid Arthritis: Results from the IORRA Observational Cohort Study

Introduction

Patients with rheumatoid arthritis (RA) have a higher prevalence of osteoporosis and hip fracture than healthy individuals. Multiple genetic loci for osteoporotic fracture were identified in recent genome-wide association studies. The purpose of this study was to identify genetic variants associated with the occurrence of hip fracture in Japanese patients with RA.

Methods

DNA samples from 2,282 Japanese patients with RA were obtained from the DNA collection of the Institute of Rheumatology Rheumatoid Arthritis cohort (IORRA) study. Six single nucleotide polymorphisms (SNPs) that have been reported to be associated with fractures in recent studies were selected and genotyped. Forty hip fractures were identified with a maximum follow-up of 10 years. The genetic risk for hip fracture was examined using a multivariate Cox proportional hazards regression model.

Results

The risk analyses revealed that patients who are homozygous for the major allele of SNP rs6993813, in the OPG locus, have a higher risk for hip fracture (hazard ratio [95% CI]  = 2.53 [1.29–4.95], P  = 0.0067). No association was found for the other SNPs.

Conclusions

Our results indicate that an OPG allele is associated with increased risk for hip fracture in Japanese patients with RA.     

The phosphatidylserine receptor TIM4 utilizes integrins as coreceptors to effect phagocytosis

T-cell immunoglobulin mucin protein 4 (TIM4), a phosphatidylserine (PtdSer)-binding receptor, mediates the phagocytosis of apoptotic cells. How TIM4 exerts its function is unclear, and conflicting data have emerged. To define the mode of action of TIM4, we used two distinct but complementary approaches: 1) we compared bone marrow–derived macrophages from wild-type and TIM4^−/− mice, and 2) we heterologously expressed TIM4 in epithelioid AD293 cells, which rendered them competent for engulfment of PtdSer-bearing targets. Using these systems, we demonstrate that rather than serving merely as a tether, as proposed earlier by others, TIM4 is an active participant in the phagocytic process. Furthermore, we find that TIM4 operates independently of lactadherin, which had been proposed to act as a bridging molecule. Of interest, TIM4-driven phagocytosis depends on the activation of integrins and involves stimulation of Src-family kinases and focal adhesion kinase, as well as the localized accumulation of phosphatidylinositol 3,4,5-trisphosphate. These mediators promote recruitment of the nucleotide-exchange factor Vav3, which in turn activates small Rho-family GTPases. Gene silencing or ablation experiments demonstrated that RhoA, Rac1, and Rac2 act synergistically to drive the remodeling of actin that underlies phagocytosis. Single-particle detection experiments demonstrated that TIM4 and β1 integrins associate upon receptor clustering. These findings support a model in which TIM4 engages integrins as coreceptors to evoke the signal transduction needed to internalize PtdSer-bearing targets such as apoptotic cells.     

Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG-7)

We have demonstrated a method for the in situ determination of the cell cycle phases of TIG-7 fibroblasts using a laser scanning cytometer (LSC) which has not only a function equivalent to flow cytometry (FCM) but also has a capability unique in itself. LSC allows a more detailed analysis of the cell cycle in cells stained with propidium iodide (PI) than FCM. With LSC it is possible to discriminate between mitotic cells and G₂ cells, between post-mitotic cells and G₁ cells, and between quiescent cells and cycling cells in a PI fluorescence peak (chromatin condensation) vs. fluorescence value (DNA content) cytogram for cells stained with PI. These were amply confirmed by experiments using colcemid and adriamycin. We were able to identify at least six cell subpopulations for PI stained cells using LSC; namely G₁, S, G₂, M, postmitotic and quiescent cell populations. LSC analysis facilitates the monitoring of effects of drugs on the cell cycle.     

Sphingolipid Biosynthesis Is Necessary for Dendrite Growth and Survival of Cerebellar Purkinje Cells in Culture

Abstract: The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 µM fumonisin B₁, a fungal inhibitor of mammalian ceramide synthase, inhibited incorporation of [³H]galactose/glucosamine and [¹⁴C]serine into complex sphingolipids of cultured cerebellar neurons. Under this condition, the expression of Purkinje cell-enriched sphingolipids, including GD1α, 9-O-acetylated LD1 and GD3, and sphingomyelin, was significantly decreased. After 2 weeks' exposure to fumonisin B₁, dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag, but their branches began to degenerate. In some cells, formation of elongated dendrite trees was severely impaired. However, treatment with fumonisin B₁ also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells, morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B₁. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed, these effects of fumonisin B₁ were reversed, but not completely, by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]caproyl]sphingosine (C₆-NBD-ceramide), a synthetic derivative of ceramide. Thus, we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells.     

Size and species-specific primary productivity and community structure of phytoplankton in Tokyo Bay

Combined methods of size fractionation and single-cell isolationwere used to investigate the seasonal variation of phytoplanktondynamics in Tokyo Bay with an emphasis on primary productivity.Red tides occurred in Tokyo Bay from spring to autumn; a diatom,Skeletonema costatum, and a raphidophycean, Heterosigma akashiwo,were the most important primary producers. Small diatoms andflagellates, including these species, were dominant and showedrapid changes of phytoplankton community structure within severaldays in summer. The nanoplankton (3–20 µm) fractioncontributed most to chlorophyll a concentration and primaryproductivity during spring to autumn, whereas the microplankton(>20 µm) contribution was remarkable in winter. Picoplankton(<3 µm phytoplankton) remained relatively constantthroughout the year. A significant reverse relationship wasobtained between assimilation rate and chlorophyll a contentfor the total and nanoplankton population; the assimilationrate was high at the initial phase of the bloom, then decreasedto a minimum level at the peak of the bloom. Factors controllingthe reduction of assimilation rates at the peak, and changesin phytoplankton community structure, are discussed.     

The MAP1-LC3 conjugation system is involved in lipid droplet formation

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.