Molecular structure of bacterial endotoxin (Escherichia coli Re lipopolysaccharide): implications for formation of a novel heterogeneous lattice structure

Analyses of crystals of Escherichia coli Re lipopolysaccharide (LPS) formed after storage in 1% triethylamine indicate that the LPS molecules are assembled to form a monolayered structure consisting of a novel heterogeneous lattice structure, the greater part of which is occupied by one kind of lattice (lattice I), corresponding to the acyl chain portion of lipid A, and the remainder is occupied by the other kind of lattice (lattice II), corresponding to the 3-deoxy-Dmanno-octulosonic acid (dOclA) dimer and the N-acetylglucosamine disaccharide of lipid A. X-ray diffraction reveals that the type of cell is monoclinic (a = 5.53 A, b = 27.2 A, c = 6.47 A, alpha = 90 degrees, beta = 125.8 degrees, gamma = 90 degrees ). Atomic force microscopy shows that crystals consist of multiple layers; the thickness of a layer corresponds to the b-axis value, and two types of surface topographies are visualized. One, regarded as the view onto the acyl chain ends, is two-dimensional arrays of oval bodies that constitute the lattice, with the lattice constants corresponding to the a- and c-axes and the angle of beta (lattice I). The other, regarded as the view onto the dOclA dimers, is two-dimensional arrays of dromedary-back-like bodies that constitute the lattice with axes of 9.0 and 10.7 A and the angle of 65 degrees formed by both axes (lattice II). Based on these results, we present the molecular model of E. coli Re LPS.     

Ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibits the growth of Escherichia coli at alkaline pH

The growth of Escherichia coli was inhibited by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in a medium of initial pH 8.8. The growth inhibition was reversed by the addition of CaCl(2). E. coli could grow in the presence of EGTA at pH values below 8. The concentration of free calcium ions increases with a decrease in medium pH because of a decrease in the calcium binding capacity of EGTA. So, although the results suggest that calcium ions are essential for the growth of E. coli, the minimum concentration required is very low.     

Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts

In bone development and regeneration, angiogenesis and bone/cartilage resorption are essential processes and are closely associated with each other, suggesting a common mediator for these two biological events. To address this interrelationship, we examined the effect of vascular endothelial growth factor (VEGF), the most critical growth factor for angiogenesis, on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. VEGF caused a dose- and time-dependent increase in the area of bone resorption pits excavated by the isolated osteoclasts, partially by enhancing the survival of the cells. Two distinct VEGF receptors, KDR/Flk-1 and Flt-1, were detectable in osteoclasts at the gene and protein levels, and VEGF induced tyrosine phosphorylation of proteins in osteoclasts. Thus, osteoclastic function and angiogenesis are up-regulated by a common mediator such as VEGF.     

Development of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with D-galactose monosaccharide: biological activities in vivo

In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1 (rhIL-1) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-1 exhibited reduced activities in vitro by 10 to 10 000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, 1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1.     

A Multicenter,Randomized, Double-Blind,Placebo-Controlled Trial of High-Dose Rebamipide Treatment for Low-Dose Aspirin-Induced Moderate-to-Severe Small Intestinal Damage

Background

Low-dose aspirin (LDA) frequently causes small bowel injury. While some drugs have been reported to be effective in treating LDA-induced small intestinal damage, most studies did not exclude patients with mild damage thought to be clinically insignificant.

Aim

We conducted a multicenter, randomized, double-blind, placebo-controlled trial to assess the efficacy of a high dose of rebamipide, a gastroprotective drug, for LDA-induced moderate-to-severe enteropathy.

Methods

We enrolled patients who received 100 mg of enteric-coated aspirin daily for more than 3 months and were found to have more than 3 mucosal breaks (i.e., erosions or ulcers) in the small intestine by capsule endoscopy. Eligible patients were assigned to receive either rebamipide 300 mg (triple dose) 3 times daily or placebo for 8 weeks in a 2:1 ratio. Capsule endoscopy was then repeated. The primary endpoint was the change in the number of mucosal breaks from baseline to 8 weeks. Secondary endpoints included the complete healing of mucosal breaks at 8 weeks and the change in Lewis score (an endoscopic score assessing damage severity) from baseline to 8 weeks.

Results

The study was completed by 38 patients (rebamipide group: n = 25, placebo group: n = 13). After 8 weeks of treatment, rebamipide, but not placebo, significantly decreased the number of mucosal breaks (p = 0.046). While the difference was not significant (p = 0.13), the rate of complete mucosal break healing in the rebamipide group (32%, 8 of 25) tended to be higher than that in the placebo group (7.7%, 1 of 13). Rebamipide treatment significantly improved intestinal damage severity as assessed by the Lewis score (p = 0.02), whereas placebo did not. The triple dose of rebamipide was well tolerated.

Conclusions

High-dose rebamipide is effective for the treatment of LDA-induced moderate-to-severe enteropathy.

Trial Registration

UMIN Clinical Trials Registry UMIN000003463     

Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca²⁺ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca²⁺-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca²⁺ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC₅₀ = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC₅₀ = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC₅₀ = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.     

Xylem water-conducting patterns of 34 broadleaved evergreen trees in southern Japan

A dye injection method was used to elucidate the xylem water-conducting pathways of 34 broadleaved evergreen trees growing in southern Japan: two semi-ring-porous, 26 diffuse-porous, five radial-porous and one non-vessel species. The large earlywood vessels in semi-ring-porous species have a water transport function in only the outermost annual ring, as in deciduous ring-porous species. On the other hand, the small vessels in semi-ring-porous species maintain the water transport function in many outer annual rings. For the other xylem-type species, the many vessels in many outer annual rings have a water transport function. In diffuse-porous species, we categorized the water-conducting pattern within the annual rings into two types: d1 type, where water travels through vessels in the whole region; and d2 type, where water travels mainly through the earlywood vessels. The pattern in radial-porous species is similar to that in the d1 type; the pattern in non-vessels species is similar to that in the d2 type. The vessel diameter in radial-porous species is similar to that of the earlywood vessels of semi-ring-porous species. These results suggest that the conduit diameter size is only one of many factors determining the water-conducting pathways of broadleaved evergreen species.     

Development of a novel fluorometric assay for nitric oxide utilizing sesamol and its application to analysis of nitric oxide‐releasing drugs

Nitric oxide (NO) is related to various physiological effects as well as to numerous diseases caused by accentuation of NO production. Measurement of NO in cells and tissues is difficult as NO readily reacts with other molecules; furthermore, its half‐life as a radical is fleeting. Currently, many NO pharmaceuticals are marketed as therapeutic agents for ischemic disease. Consequently, the identification of NO radicals and determination of generation rate from pharmaceuticals is very important when the effect of the medicinal supply is estimated. In this study, we developed a fluorometric assay for NO employing sesamol (3,4‐methylenedioxyphenol) as a fluorometric substrate. Sesamol is converted to a fluorescent derivative (ex. 365 nm, em. 447 nm), which is dimmer in the presence of NO. The detection limit of NO with this method is 400 fmol; moreover, NO generated from drugs can be measured. Copyright © 2009 John Wiley & Sons, Ltd.