Preparation of a topologically linked branch polymer containing cyclodextrin

Macromolecules containing topological linkages made of α‐cyclodextrin (αCD) and poly(ethylene glycol) (PEG) were prepared by condensation of mono‐6‐O‐deoxy‐mono‐6‐amino‐α‐cyclodextrin (NH₂‐αCD) with PEG dicarboxylic acid (PEG‐diCOOH) having one carboxyl group at both chain ends. The analysis of ¹H NMR and ¹³C NMR spectra of the condensation products showed completion of the condensation reaction between NH₂ and COOH groups, and the absence of ester linkages, thus indicating that all of the PEG chains carried one αCD molecule at both chain ends through amide linkages. Gel permeation chromatography analysis of these condensation products showed that NH₂‐αCD formed inclusion complexes with PEG‐diCOOH prior to condensation, resulting in macromolecules having topological linkages. In addition, the amount of the topological linkages increased with the increase of molecular weight of PEG‐diCOOH. This result shows that the complexation equilibrium of NH₂‐αCD with PEG‐diCOOH depends on the concentration of ethylene glycol units. Copyright © 2007 Society of Chemical Industry     

Monoclonal antibody specific to bovine lens epithelial cell membrane: preparation of a lens epithelial cell-selective immunotoxin

Dithranol has been used successfully in the treatment of psoriasis for more than 75 years, and much in vitro and in vivo research has been done on the elucidation of the mode of action of this potent and safe antipsoriatic therapy. In vivo research has revealed major effects of dithranol on epidermal proliferation and inflammation. Information on the in vivo effects on epidermal differentiation is limited. Therefore, the dynamics of a set of differentiation markers (keratin 16, filaggrin, keratinocyte transglutaminase, involucrin) and markers for proliferation and inflammation (Ki-67, T lymphocytes, polymorphonuclear leucocytes) were studied in skin biopsies of six patients with psoriasis during 4 weeks of dithranol therapy. The treatment regimen involved a short contact protocol at our out-patient day treatment centre with an easily washed off cream. Treatment resulted in a decrease of the PASI score of 48% in 4 weeks. Immunohistochemically, a major decrease of keratin 16 content and virtually complete restoration of the filaggrin positive cell layer were seen. These changes proved to be significant by comparison of the markers over the group of six patients. Although many other topical treatments for psoriasis (occlusive therapy and vitamin D3 analogues) result in a prominent reduction in the amount of transglutaminase and involucrin positive cell layers, the effect of dithranol on these markers is minimal.     

Direct observation of the separation process by the chromato-videoscope

Separation process in a liquid chromatographic column were visualized and analyzed by a developed chromato-videoscope. The migration aspects were evaluated with successively obtained densitograms. In reversed-phase chromatography, the band widths of each solute band were almost equal fore both weakly and strongly retained solutes when compared at the same column position (the same migration distance in the column). In the gradient elution mode, the position of the solute band showed that the migration velocity of the solute band changed gradually according to changes in solvent composition. The drug trapping process to BSA- coated ODS packings for direct injection of biological fluid was also observed. In the absence of BSA from the sample solution, the drug molecules were trapped in a narrow band. However, at higher BSA concentrations in the sample solution, a broader band shape was observed. This band broadening shows how the drug molecules were retained on the protein.     

Scanning electron microscopic characteristics of small-incision intraocular lenses

PURPOSE: To evaluate the surface characteristics of commonly used, small-incision, intraocular lenses (IOLs). METHODS: Representative samples of five groups of foldable IOLs (4 silicone and 1 acrylic) underwent surface and edge-finish examination using a slit lamp. The IOLs were folded using a folding block and forceps. All the IOLs then were examined using a scanning electron microscope. A one-piece polymethylmethacrylate IOL was used for comparing surface-finish characteristics. The IOLs were examined for optic surface quality, edge finish, haptic, haptic/optic junction, and possible post-folding modifications. RESULTS: Slit-lamp evaluation of the surface quality of all of the silicone lenses demonstrated a smooth finish of the optic surface, edge, and haptics. Scanning electron microscopic analysis of the IOLs demonstrated adequately finished haptics or footplates and optics. Excess molding flash was seen on the edges of the some of the silicone IOLs, and no molding flash was observed on others. The acrylic IOL had a somewhat sharper optic edge. Irregular finish of the haptic/optic junctions of some of the IOLs (both silicone and acrylic) was noted. CONCLUSIONS: Currently available foldable IOLs have demonstrated adequate lens finish. However, irregularities of the haptic/optic junctions and molding flash are present on most IOLs evaluated, indicating room for improvement in the finish of foldable IOLs. Phacoemulsification with capsular bag IOL placement may decrease the clinical significance of these relatively subtle lens finish irregularities.     

Endoscopic mucosal resection for early esophageal cancer]

OBJECTIVES: We investigated the role of endogenous endothelin-1 in the development of cardiac hypertrophy in vivo under pressure overload conditions. BACKGROUND: Endothelin-1, a potent vasoconstrictor peptide, has recently been shown to act as a growth factor of myocardial cells in culture. METHODS: We examined the effect of an endothelin-A receptor antagonist (FR139317) on the development of right ventricular hypertrophy in rats with monocrotaline-induced pulmonary hypertension. Three groups of rats were studied: those given monocrotaline alone or monocrotaline plus FR139317 and those given vehicle alone (control group). RESULTS: The ratio of right ventricular systolic pressure to aortic systolic pressure was similarly elevated in rats treated with monocrotaline and monocrotaline plus FR139317. The right ventricular/left ventricular weight ratio was increased in monocrotaline-treated rats but lower in rats treated with monocrotaline plus FR139317 than in those treated with monocrotaline alone (p < 0.01). As a biochemical marker of hypertrophy, the isoform ratio of beta-myosin heavy chain protein was determined for the right ventricular tissue samples. This ratio was increased in all monocrotaline-treated rats but was lower (p < 0.01) in rats given monocrotaline plus FR139317 than in those given monocrotaline alone. The isoform ratio of beta-myosin heavy chain messenger ribonucleic acid quantitated by S1 nuclease mapping also was lower (p < 0.025) in rats receiving monocrotaline plus FR139317 than in those receiving monocrotaline alone. CONCLUSIONS: These data suggest that blocking the action of endothelin-1 with a receptor antagonist ameliorates cardiac hypertrophy in this model system, and that this action is not mediated by ameliorating hemodynamic changes.     

Effects of a hydroxyl radical scavenger on delayed ischemic neurological deficits following aneurysmal subarachnoid hemorrhage: results of a multicenter, placebo-controlled double-blind trial

A water-soluble, novel synthetic compound, AVS ((+/-)-N, N'-propylenedinicotinamide; nicaraven) has no demonstrable vasoactive properties but scavenges hydroxyl radicals in aqueous environmental conditions at neutral pH. Based on the results of preceding experimental and clinical studies showing marked ameliorative effects of AVS on cerebral vasospasm and ischemic brain damage, a multicenter, placebo-controlled double-blind clinical trial was undertaken to verify its beneficial effects on delayed ischemic neurological deficits (DINDs) due to vasospasm and on the overall outcome of patients with subarachnoid hemorrhage (SAH). A total of 162 patients with SAH who had Glasgow Coma Scale scores between 7 and 15 on admission were enrolled in the trial. Drug administration (4 g AVS or 4 g glucose as placebo; infused intravenously for 6-8 hours once a day) was begun within 5 days post-SAH and continued for 10 to 14 days. Intent-to-treat analysis of these patients revealed that the overall incidence of DINDs, which was defined as an exacerbation of impaired consciousness and/or focal neurological deficits, was significantly reduced, by 34.5% (placebo 54.2%, AVS 35.5%; p < 0.05, Mann-Whitney U-test). The Glasgow Outcome Scale (GOS) score at 1 month was significantly improved by AVS (p < 0.05, U-test). At 3 months, the difference in the GOS scores between the groups became marginal on U-tests (p < 0.10), but the percentage of good outcome tended to increase, with a relative increase of 20.3% (AVS 76.3%, placebo 63.4%; p < 0.10, chi-square test), and the cumulative incidence of death was significantly reduced (p < 0.05, log-rank test). No significant adverse reaction attributable to treatment was observed. the usefulness of AVS in therapy for SAH is strongly indicated by the fact that the agent significantly ameliorated DINDs, leading to a marked improvement in the GOS scores at 1 month, as well as a reduction in the cumulative incidence of death by 3 months.     

Long-chain acyl-CoA hydrolase from rat brain cytosol: purification, characterization, and immunohistochemical localization

To determine whether scavenger receptors are susceptible to regulation by granulocyte macrophage colony-stimulating factor (GM-CSF), a macrophage-specific cytokine, human monocytes were differentiated into macrophages in the absence or presence of 20 U/mL GM-CSF. Binding, uptake, and degradation of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) were measured. Treatment with GM-CSF resulted in a significant twofold to threefold decrease in the number of binding sites for Ac-LDL and Ox-LDL on the surface of macrophages without affecting the affinity of the receptor for these ligands. Competition experiments revealed that two binding sites were responsible for the recognition and uptake of Ac-LDL; one specific for Ac-LDL and one that recognized both Ac-LDL and Ox-LDL. No binding site specific for Ox-LDL could be detected in either control or GM-CSF-treated macrophages. Treatment of human monocyte-derived macrophages with GM-CSF resulted in a decrease of the Ac-LDL/Ox-LDL receptor but did not affect the binding site specific for Ac-LDL. Northern blot analysis showed that mRNA levels of both types I and II scavenger receptor were reduced in macrophages differentiated in the presence of GM-CSF. Human macrophages that were differentiated in the presence of GM-CSF accumulated approximately 50% fewer cholesteryl esters. Taken together, these results indicate that GM-CSF can downregulate both types I and II scavenger receptor in human monocyte-derived macrophages, which might have implications for foam cell formation.     

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.     

Mitochondrial targeting of glutathione reductase requires a leader sequence

Glutathione reductase (GR), which catalyzes the conversion of glutathione disulfide to glutathione, is encoded in nuclear DNA, but is active in cytoplasm and mitochondria. However, analyses of known protein and DNA sequences for human GR have not revealed a potential mitochondrial targeting signal (MTS). We generated two 5'-truncated GR clones, which resulted in omission of the N-terminal 5 or 10 amino acids, to disable a potential targeting signal, and generated two GR clones containing synthetic MTS cDNAs. Transfection of Chinese hamster ovary cells with the full length human GR cDNA or with the 5'-truncated clones increased cytosolic GR activities 6- to 14-fold, but increased mitochondrial activities less than 2-fold. In contrast, transfection with either of the GR clones containing MTS cDNAs increased GR activities in mitochondria more than 24-fold. We conclude that the existing protein and DNA sequences for human GR do not contain a MTS and that such a signal is needed for effective mitochondrial targeting.     

Osteoclastic resorption of bone-like apatite formed on a plastic disk as an in vitro assay system

Congenital diaphragmatic hernia (CDH) is associated with high neonatal mortality from lung hypoplasia and persistent pulmonary hypertension. Pulmonary neuroendocrine cells (PNEC) produce calcitonin gene-related peptide (CGRP), a potent vasodilator. We previously reported altered distribution of CGRP-positive PNEC in full-term rats with CDH, that may lead to an imbalance in vasoactive mediators. In the present study we examined the expression of CGRP-positive PNEC during lung development in rats with CDH induced by 2,4-dichlorophenyl-p-nitrophenylether (Nitrofen). Cesarean sections were performed on Days 16, 18, 20, or 22, and the lungs were immunostained for CGRP and immunoreactive cells were quantitated through image analysis. On Day 16, CGRP-immunoreactive staining was negative; on Day 18, CGRP-immunoreactive cells were found in all controls (not exposed to Nitrofen), whereas in CDH pups, CGRP-positive cells were present in only four of six cases. On Day 20, CGRP immunoreactivity was similar in CDH pups, Nitrofen-exposed pups without CDH, and controls. On Day 22 (term), significantly more CGRP-positive cells (i.e., number of positive cells per surface area [mm2] or lung volume [mm3]) were found in ipsilateral lungs of CDH pups than in controls (P < 0.05). The difference was even more striking in contralateral lungs of CDH pups (P < 0.001), ruling out nonspecific effects of Nitrofen. In CDH lungs, the proportion of immunostained epithelium and the size of the neuroendocrine cell clusters (neuroepithelial bodies [NEB]) were not significantly different from those of controls. On Day 22, supraoptimal dilution immunocytochemistry yielded similar results in CDH pups and controls. We conclude that in CDH, CGRP expression in PNEC and NEB is delayed during early stages of lung development. Because CGRP also exhibits growth factor-like properties for endothelium and epithelial cells, the lack of this factor during a crucial developmental stage (canalicular period) may be causally related to lung hypoplasia.