Enhanced responses of glycosylphosphatidylinositol anchor-deficient T lymphocytes

The functions of GPI-anchored proteins in T lymphocyte activation have been controversial. This issue was addressed by studying the responses of T lymphocytes from T lymphocyte-specific GPI anchor-deficient mice to different stimuli that normally allow coligation of TCR and GPI-anchored proteins. Stimulation of GPI anchor-deficient T lymphocytes with ConA induced 2-fold higher proliferative responses than did normal cells. In response to allogeneic stimulation, proliferation of GPI anchor-deficient T lymphocytes was enhanced 2- to 3-fold. The response to ConA of a GPI anchor-deficient anti-OVA T lymphocyte clone generated from these mice was approximately 3-fold higher than that of cells from the same clone in which GPI anchor expression was restored by retroviral transduction. The response of the GPI anchor-deficient cloned anti-OVA T lymphocytes to antigenic stimulation was similar to that of the retrovirally restored cells. These results indicate that coligation with GPI-anchored proteins counteracts the response to TCR stimulation by ConA or alloantigen but not protein Ag.     

Expression of HSP 70 and its mRNAS during ischemia-reperfusion in the rat bladder

HSP 70 is an important protein that repairs damaged tissue after injury. In the present study, we investigated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder dome. Male Wistar rats, 8 weeks old, were divided into six groups: controls, 30-min ischemia, 30-min ischemia and 30-, 60-minute, 1- and 7-day reperfusion, groups A, B, C, D, E, and F, respectively. In functional studies, contractile responses to carbachol were measured in these groups. The expression of HSP 70-1/2 mRNAs was quantified using a real-time PCR method, and that of HSP 70 proteins was measured using ELISA in the bladders. In the functional study, Emax values of carbachol to bladders in the A, B, C, D, E and F groups were 9.3 +/- 1.3, 7.9 +/- 1.7, 4.3 +/- 0.8, 4.2 +/- 0.7, 4.5 +/- 0.6, and 8.1 +/- 1.2 g/mm2, respectively. In the control group, the expression of HSP 70-1/2 mRNA was detected, and the expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1/2 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70 proteins was increased shortly after the expression of their mRNAs. The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels. Our data indicate that contractile responses of the bladder were decreased by ischemia reperfusion, and that expression of HSP 70 and its mRNAs appeared to increase after a short period of the insult.     

Transforming growth factor-alpha directly augments histidine decarboxylase and vesicular monoamine transporter 2 production in rat enterochromaffin-like cells

For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-alpha (TGF-alpha) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-alpha during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-alpha and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-alpha mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-alpha stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-alpha in vivo and in vitro. During gastric mucosal injury, TGF-alpha was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression.     

Age-related changes of the ultrastructure in the cardiomyopathic hamster (UM-X7.1 Syrian hamster) parathyroid gland

We qualitatively and quantitatively investigated parathyroid glands of the UM-X7.1 cardiomyopathic hamster at 1, 2, 6 and 12 months of age to compare them with those of the normal hamster. We found that at 1 month of age in the UM-X7.1 hamster, the Golgi apparatus, lipid droplets and secretory granules decreased. There were no significant differences between the UM-X7.1 hamster and the control hamster at 2 months of age. At 6 months of age, the Golgi apparatus, rER and the secretory granules significantly increased in the UM-X7.1 hamster. At 12 months of age, the Golgi apparatus and lysosomes increased, while the secretory granules decreased. Ultrastructurally, we consider that in the UM-X7.1 hamster, the synthesis and release of the parathyroid at 6 months of age may be activated by an excessive amount of circulating catecholamine, and the functional activity of the parathyroid glands at 12 months of age may be depressed by the increased plasma calcium level. These findings suggest that the activities of the synthesis and release of the parathyroid hormone were the highest at 6 months of age in the UM-X7.1 hamster.     

Cloning and characterization of a gene encoding the γ-butyrolactone autoregulator receptor from <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>

With primers designed for the conserved region of the -butyrolactone autoregulator receptor proteins from Streptomyces species, PCR using the Streptomyces clavuligerus genome DNA as a template gave a clear band of 100 bp, the sequence of which revealed high similarity to the expected region of a receptor gene. By Southern blot and colony hybridization with the 100-bp insert as a probe, plasmid pSCA, harboring a 4.2 kb-SalI fragment, was obtained. Sequence analysis on the insert revealed a 702-bp ORF encoding a protein with a moderate similarity (identity, 33–43%; similarity, 51–62%) to known -butyrolactone autoregulator receptor proteins from Streptomyces sp. The ORF was named scaR (S. clavuligerus autoregulator receptor). The scaR/pET-3d plasmid was constructed for overexpression of the recombinant ScaR protein (rScaR) in Escherichia coli, and the rScaR protein was purified to homogeneity by DEAE-ion-exchange HPLC. The molecular mass of the purified rScaR protein was determined to be 27 kDa as determined by SDS-PAGE, and 54 kDa by gel filtration HPLC under nondenatured conditions at a low protein concentration, indicating that the majority of the native ScaR is present in the form of a dimer, although rScaR tended to aggregate into a higher molecular form of 230 kDa at a high protein concentration. A binding assay with tritium-labeled autoregulators indicated that IM-2 type compounds with a long C2 side chain were the most effective ligands for rScaR, demonstrating for the first time that the -lactam producer S. clavuligerus contains a gene for the -butyrolactone autoregulator receptor.     

Contribution of root cap mucilage and presence of an intact root cap in maize (Zea mays) to the reduction of soil mechanical impedance

BACKGROUND AND AIMS: The impedance to root growth imposed by soil can be decreased by both mucilage secretion and the sloughing of border cells from the root cap. The aim of this study is to quantify the contribution of these two factors for maize root growth in compact soil. METHODS: These effects were evaluated by assessing growth after removing both mucilage (treatment I -- intact) and the root cap (treatment D -- decapped) from the root tip, and then by adding back 2 micro L of mucilage to both intact (treatment IM -- intact plus mucilage) and decapped (treatment DM -- decapped plus mucilage) roots. Roots were grown in either loose (0.9 Mg m(-3)) or compact (1.5 Mg m(-3)) loamy sand soils. Also examined were the effects of decapping on root penetration resistance at three soil bulk densities (1.3, 1.4 and 1.5 Mg m(-3)). KEY RESULTS: In treatment I, mucilage was visible 12 h after transplanting to the compact soil. The decapping and mucilage treatments affected neither the root elongation nor the root widening rates when the plants were grown in loose soil for 12 h. Root growth pressures of seminal axes in D, DM, I and IM treatments were 0.328, 0.288, 0.272 and 0.222 MPa, respectively, when the roots were grown in compact soil (1.5 Mg m(-3) density; 1.59 MPa penetrometer resistance). CONCLUSIONS: The contributions of mucilage and presence of the intact root cap without mucilage to the lubricating effect of root cap (percentage decrease in root penetration resistance caused by decapping) were 43 % and 58 %, respectively. The lubricating effect of the root cap was about 30 % and unaffected by the degree of soil compaction (for penetrometer resistances of 0.52, 1.20 and 1.59 MPa).     

Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A-dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.     

A comparison of the ability of XMAP215 and tau to inhibit the microtubule destabilizing activity of XKCM1

During mitosis, microtubules not only grow fast, but also have a high rate of catastrophe. This is achieved in part by the activity of the MAP, XMAP215, which can stimulate the growth rate of microtubules without fully inhibiting the function of the catastrophe-kinesin XKCM1. We do not know whether this activity is particular to XMAP215, or is a general property of all MAPs. Here, we compare the activities of XMAP215 with the neuronal MAP tau, in opposing the destabilizing activity of the non-conventional kinesin XKCM1. We show that tau is a much more potent inhibitor of XKCM1 than XMAP215. Because tau completely suppresses XKCM1 activity, even at low concentrations, the combination of tau and XKCM1 is unable to generate mitotic microtubule dynamics.