Caveolin-1 regulates the functional localization of N-acetylglucosaminyltransferase III within the golgi apparatus

In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core beta-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density gradient ultracentrifugation. Caveolin-1 may modify the biosynthetic pathway of sugar chains via the regulation of the intra-Golgi subcompartment localization of this key glycosyltransferase.     

Serum paraoxonase activity decreases in rheumatoid arthritis

OBJECTIVE: To estimate the alterations of paraoxonase 1 (PON1) and high-density lipoprotein (HDL) in rheumatoid arthritis (RA). DESIGN AND METHODS: We investigated the serum enzyme activity and concentration of PON1 and their relationship with serum lipids, high-density lipoprotein (HDL) parameters, and acute phase reactants of serum amyloid A (SAA) and C-reactive protein (CRP) in patients with RA. RESULTS: Serum paraoxonase (PON) activity was significantly decreased in RA patients (n = 64, 131 +/- 53 micro mol/min/L) compared with healthy subjects (n = 155, 164 +/- 59) despite the absence of any difference in serum lipid levels between the two groups. This decrease of serum PON activity in RA patients was found in every genotype (Q/Q, Q/R, R/R) of PON1 at 192 Q/R. There was a different distribution in PON1 Q/R genotypes between RA patients and healthy subjects, and RA patients exhibited less (44%) positive PON1-Q than did the healthy subjects (66%). In a further investigation of age- and gender-matched subgroups of RA (n = 25) and healthy subjects (n = 25), not only serum PON activity, but also lecithin-cholesterol acyltransferase (LCAT) was found to be significantly decreased in RA patients (125 +/- 61 micro mol/min/L, 63.2 +/- 17.2 nmol/ml/hr/37 degrees C) than in healthy subjects (169 +/- 67, 74.7 +/- 19.5), respectively. PON1 and LCAT as well as HDL constituent apolipoprotein (apo) AI and apo AII, were altered significantly in RA patients. CONCLUSIONS: Acute-phase HDL, which is remodeled structurally and functionally in RA, might be less anti-atherogenic due to the impairment of original HDL function. These alterations of HDL in RA patients may explain in part the reported increase in cardiovascular mortality in patients with RA.     

Electrostatic guidance of glycosyl cation migration along the reaction coordinate of uracil DNA glycosylase

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2'-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion-uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.     

Powering DNA repair through substrate electrostatic interactions

The reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K(m) for the series of MeP-substituted substrates against log K(D) for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR(+), and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA.     

Effects of ionic valency of interacting metal elements in ion uptake by carrot (Daucas carota cv. U.S. harumakigosun)

Interaction of elements in the course of element uptake by carrot (Daucas carota cv. U.S. harumakigosun) exerted by the addition of elements, such as Rb, Zn, and Al, was investigated. For the purpose of precise evaluation of uptake behavior, the simultaneous determination of absorption of Na, Be, Sr, Mn, Co, Zn, Ce, Pm, and Gd was conducted by the multitracer technique. For root uptakes, Al exhibited its influence on the uptake of essential elements and on the uptake of toxic or unbeneficial ones, presumably as a result of the large electric valency that caused cell membrane disintegrity. On the other hand, Zn as a divalent cation only affected the uptake of essential and beneficial elements. Rubidium, which is a monovalent cation, did not exhibit any effect on the uptake of other ions. Concerning shoot uptakes, inhibition by Zn and Al, but not by Rb, was observed for the uptake of Sr, Mn, Co, and Zn. From the present investigation, it is suggested that there exists an interaction between added ions and the elements taken into plants and that the degree of interaction increases in the increasing order of ionic valency: M+ (Rb), M2+ (Zn), and M3+ (Al).     

Increased expression of cardiotrophin-1 during ventricular remodeling in hypertensive rats

Cardiotrophin-1 (CT-1) stimulates longitudinal myocardial cell hypertrophy. We examined the expression of CT-1, leukemia inhibitory factor (LIF), and gp130 by competitive RT-PCR and Western blotting in Dahl salt-sensitive (DS) rats with a high-salt diet, which showed a distinct transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF). The expression levels of CT-1 mRNA and protein were significantly increased at the CHF stage compared with the LVH stage and age-matched Dahl salt-resistant (DR) rats (n = 6 for each group). mRNA expression of LIF was not changed in the left ventricle at any stage by RT-PCR. gp130 mRNA and protein levels of DS rats at 11 and 17 wk were significantly increased compared with age-matched DR rats. The isolated myocyte length of DS rats at 17 wk was the longest among the four groups of rats. The LV end-diastolic dimension (LVDd) of DS rats, determined by echocardiography, was significantly increased at the CHF stage. There was a significant correlation between the CT-1 protein level and LVDd. CT-1 may play a role in ventricular remodeling during transition from LVH to CHF in the rat hypertensive model.     

Foot formation in hydra: commitment of the basal disk cells in the lower peduncle

Foot regeneration in the freshwater hydra Pelmatohydra robusta was examined using a monoclonal antibody AE03 as a marker. This antibody specifically recognizes mucous-producing ectodermal epithelial cells in the basal disk, but not cells in the peduncle region located just above the basal disk in the foot. When the basal disk was removed by amputation at the upper or lower part of the peduncle, AE03-positive (basal disk) cells always appeared at the regenerating tip of the footless polyp approximately 12-16 h later. When a small piece of tissue was cut out from the upper or lower peduncle region, the tissue invariably turned into a smooth spherical or oblong shape within a few hours. AE03 signal appeared in these spheres variably depending on their origin: when tissue pieces were derived from the lower peduncle, the signal appeared in nearly all pieces and often covered the entire surface of the pieces within 24 h. In contrast, the signal appeared in less than 10% of pieces derived from the upper peduncle. Furthermore, the signal seldom covered more than half of the surface of these pieces. When maintained for many days, pieces derived from the upper peduncle often regenerated tentacles, whereas those from the lower peduncle seldom did. These and other observations suggest that epithelial cells in the peduncle can rapidly differentiate into basal disk cells when the basal tissue is removed. However, cells in the upper peduncle are not irreversibly committed to differentiate into basal disk cells because, when cut out as small tissue pieces, they could remain AE03 negative and become tentacle cells. In contrast, the cells in the lower peduncle apparently are irreversibly committed to differentiate into basal disk cells, as they always turned rapidly into AE03-positive cells once they were physically separated from (and freed from the influence of) the basal disk itself, regardless of the separation methods used.