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PubMed2000

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall.

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.

Aspergillus fumigatus↗
PubMed1997

Stromal fibroblasts are associated with collagen IV in scar tissues of alkali-burned and lacerated corneas.

PURPOSE: Corneal wound healing frequently leads to the formation of opaque scar tissue. We examined whether stromal fibroblastic cells of injured corneas express collagen IV and contributes to the formation of a basal lamina-like structure. METHODS: Rabbits were anesthetized, and central corneal alkali burn (8 mm in diameter; 1 M NaOH, 1 min) or laceration (8 mm long) were produced. The injured corneas, which had healed for 1, 7, 21 and 45 days, were subjected to histological and immunohistochemical studies with goat anti-collagen IV antibodies, using light and electron microscopy, and in situ hybridization with an antisense digoxigenin-labeled riboprobe of collagen alpha 1(IV) mRNA. For comparison, twenty-day-old fetal corneas were subjected to immunohistochemical study and transmission electron microscopy (TEM). RESULTS: TEM examinations revealed that the stromal collagenous matrix was organized in orthogonal lamellae during corneal development, whereas that of alkali-burned cornea, which had healed for 3 weeks, was disorganized. The stroma of twenty-day-old fetal cornea was not labeled by the anti-collagen IV antibodies. In contrast, one week after injury, specific collagen IV immunostaining was detected in the injured stroma. As the healing proceeded (21-45 days), the antibodies reacted with fibroblastic cells and the extracellular matrix of scar tissues located in the anterior portion of alkali-burned corneas, as well as the posterior portion of lacerated corneas. The middle portion of the stromal tissues was weakly labeled by the anti-collagen IV antibodies with the exception of the blood vessel wall. Immuno-electron microscopic study showed that collagen IV and fibronectin were closely associated with the fibroblastic cells. In situ hybridization demonstrated that epithelial and endothelial cells and fibroblastic cells in the wounded corneal stroma and retro-corneal membrane expressed alpha 1(IV) mRNA, whereas in normal corneas the expression of alpha 1(IV) mRNA was limited to epithelial and endothelial cells. CONCLUSIONS: The enhanced expression of collagen IV by the fibroblastic cells in the stroma of injured corneas is consistent with the notion that they may contribute to the formation of basal lamina-like structures in injured corneas.

Animals↗
PubMed2001

Optimal concentration of human epidermal growth factor (hEGF) for epithelial healing in experimental corneal alkali wounds.

PURPOSE: By using both in vivo and in vitro (organ-cultured) systems, the optimal concentrations of hEGF to enhance epithelial healing after alkali wounds were evaluated in the rabbit cornea. METHODS: Alkali-injured corneas (pi = 5.5 mm, 1 N NaOH, 60 s) were treated with 0.01, 0.1, 1.0, 10 and 100 ng/ml hEGF for the in vitro study. The healing of epithelium and endothelium was determined at 1, 2, 3, 4, and 6 days after treatment. For the in vivo experiment, the eyes were treated with 2, 5, 10, and 50 microg/ml hEGF 3 times per day. The measurement of epithelial healing rate, transmission electron microscopy and immunohistochemical observation were performed after 7 days treatment. RESULTS: In in vitro tests, hEGF enhanced the epithelial healing rates, showing a maximum enhancement at the concentration of 1.0 ng/ml, and endothelial healing was increased at 100 ng/ml. In in vivo studies, no significant difference was observed in the rates of epithelial healing between control and each hEGF-treated group. Among the tested concentrations, 5 microg/ml hEGF induced the most active proliferation of basal cells and 50 microg/ml hEGF remarkably produced a vascular ingrowth to the central wound area. The thickness of re-surfaced epithelium was increased by hEGF in a concentration-dependent manner. CONCLUSIONS: The results of the present study indicate that a low concentration of hEGF may selectively enhance epithelial healing without affecting endothelial healing. The optimal concentration of hEGF for the stimulation of epithelial healing appears to be 5 microg/ml in rabbit corneal alkali wounds.

Animals↗
PubMed1987

The distribution of intracellular alkali metals in Reference Man.

A review of recent literature and a re-analysis of data used in Publication 23 of the International Commission on Radiological Protection (ICRP) have revealed several problems regarding the distribution of the predominantly intracellular alkali metals potassium, rubidium and caesium in ICRP Reference Man. In this paper we re-evaluate the distribution of the intracellular alkali metals in humans and we offer an approach for selecting reference concentrations for the organs of a typical adult human. For non-skeletal tissues, reference values for K, Rb and Cs are determined as a trimmed weighted mean of reported values remaining after studies involving apparently questionable populations or measurement techniques have been eliminated. The skeleton is treated as a special case, with explicit consideration being given to the heterogeneous distribution of these elements among different regions and different types of skeletal tissue. The most significant suggested changes for the intracellular alkali metals are for Rb in the skeleton and total body. ICRP Reference Man has a total Rb content of 0.68 g, compared with our estimate of only 0.25 g, and a skeletal Rb content of 0.21 g, compared with our estimate of only 0.013 g. This has important dosimetric implications for radiorubidium, because the retention model for Rb given in ICRP Publication 30 assigns a large portion of an intake to the skeleton on the basis of the large skeletal Rb content assigned to Reference Man in ICRP Publication 23.

Body Composition↗
PubMed1982

Fetal hemoglobin: optimum conditions for its estimation by alkali denaturation.

Alkali denaturation is the most commonly used technic to estimate fetal hemoglobin in red blood cells. The Betke and colleagues method (Nature 184: 1959; 1877-78) using cyanmethemoglobin (HiCN) was recommended by an International Committee for Standardization in Hematology for fetal hemoglobin levels between 2 and 40%. We showed that precision with samples containing up to 5% fetal hemoglobin can be considerably improved by measuring the absorbance of HiCN at 420 nm rather than at 540 nm because the molar absorptivity is 10 times greater in the Soret band. To determine optimum conditions for the assay, we studied the kinetics and stoichiometry of conversion of hemoglobin to HiCN with various concentrations and mixtures of ferricyanide, kinetics of alkali denaturation, ammonium sulfate precipitation of denatured HiCN, filtration as opposed to centrifugation for separating denatured HiCN, stability of HiCn in water and KCN solutions, and linearity of absorbance in the Soret band. With our modified procedure, the CV with normal amounts (less than 1%) of fetal hemoglobin improved from 28% to 8.7%. The CVs with 6.8% and 55% fetal hemoglobin were 7.0-4.4% respectively, and linear estimates were obtained with cells containing up to 50% fetal hemoglobin. We conclude that our modified alkali denaturation procedure yields reliable and reproducible estimates of fetal hemoglobin over a wide range of concentrations.

Adult↗
PubMed1980

Repair of ethylnitrosourea-induced DNA damage in the newborn rat. I. Alkali-labile lesions and in situ breaks.

The extreme sensitivity of the developing rat brain to tumor induction by N-ethyl-N-nitrosourea (ENU) has been ascribed to the relatively inefficient repair of the presumed promutagenic lesion O6-ethylguanine from brain DNA. We have compared the brain of the newborn rat with liver, kidney and lung with respect to the repair of other types of DNA lesions that ENU induces, namely single strand breaks and alkali-labile lesions. The induction and repair or loss of these lesions has been analysed by alkaline sucrose gradient sedimentation using both mild as well as strong alkaline hydrolysis conditions. We found that ENU induces few lesions in the DNA of various organs that are detectable as breaks after mild alkaline hydrolysis. 24 h after ENU treatment such lesions are no longer detectable in brain DNA, while they are detectable in the DNA from other organs up to 10 days after ENU treatment. The number of ENU-induced lesions, detectable as breaks after strong alkaline hydrolysis, is far larger. These lesions were persistent in kidney DNA, disappeared slowly from liver DNA (t(1/2) = about 10 days), and more rapidly from brain and lung DNA (t(1/2) = 2-3 days). The latter result seems to be in contradiction with various reports that a large fraction of the ENU-induced alkali-labile lesions are stable in vivo. This difference between our results and those of others might be due to a difference between proliferating and non-proliferating cells. Whether the alkali-labile lesions are removed from brain and lung DNA by a specific repair mechanism or by other causes remains to be investigated.

Adenine↗
PubMed1985

Defective repair of a class of 4NQO-induced alkali-labile DNA lesions in xeroderma pigmentosum complementation group A fibroblasts.

Normal human or xeroderma pigmentosum complementation group A (XP-A) fibroblasts were exposed to various concentrations of either 4-nitroquinoline 1-oxide (4NQO) or its 3-methyl derivative, and the rates of repair of the alkali-labile lesions induced in DNA by each agent were monitored over a period of 24 h post-treatment incubation. The data indicate that 4NQO induces at least two major classes of alkali-labile lesions into human DNA; one class disappears rapidly from the DNA of both normal and XP-A fibroblasts, while the other class undergoes repair at a relatively slow rate in normal cells, but is not removed at all in the excision-deficient cells. Methylation of 4NQO at the 3-position appears to abolish the induction of the latter class of alkali-labile lesions, whereas the rapidly removed lesions are still being induced.

4-Hydroxyaminoquinoline-1-oxide↗
PubMed2004

Alkali grass resists salt stress through high [K+] and an endodermis barrier to Na+.

In order to understand the salt-tolerance mechanism of alkali grass (Puccinellia tenuiflora) compared with wheat (Triticum aestivum L.), [K(+)] and [Na(+)] in roots and shoots in response to salt treatments were examined with ion element analysis and X-ray microanalysis. Both the rapid K(+) and Na(+) influx in response to different NaCl and KCl treatments, and the accumulation of K(+) and Na(+) as the plants acclimated to long-term stress were studied in culture- solution experiments. A higher K(+) uptake under normal and saline conditions was evident in alkali grass compared with that in wheat, and electrophysiological analyses indicated that the different uptake probably resulted from the higher K(+)/Na(+) selectivity of the plasma membrane. When external [K(+)] was high, K(+) uptake and transport from roots to shoots were inhibited by exogenous Cs(+), while TEA (tetraethylammonium) only inhibited K(+) transport from the root to the shoot. K(+) uptake was not influenced by Cs(+) when plants were K(+) starved. It was shown by X-ray microanalysis that high [K(+)] and low [Na(+)] existed in the endodermal cells of alkali grass roots, suggesting this to be the tissue where Cs(+) inhibition occurs. These results suggest that the K(+)/Na(+) selectivity of potassium channels and the existence of an apoplastic barrier, the Casparian bands of the endodermis, lead to the lateral gradient of K(+) and Na(+) across root tissue, resulting not only in high levels of [K(+)] in the shoot but also a large [Na(+)] gradient between the root and the shoot.

Biological Transport↗
PubMed1993

Effect of doxycycline hyclate on corneal epithelial wound healing in the rabbit alkali-burn model. Preliminary observations.

We examined the effects of doxycycline hyclate on epithelial healing in vivo in the rabbit alkali-burn model. Twelve 2-3-kg Dutch belted rabbits were divided into three groups and received standard bilateral alkali burns (1 N sodium hydroxide for 30 s in an 11-mm circular plastic well). In group 1, two rabbits (four eyes) served as untreated controls. In group 2, five rabbits (10 eyes) received doxycycline hyclate (1.5 mg/kg) orally daily for 14 days. In group 3, five rabbits (10 eyes) received doxycycline hyclate (5 mg/kg) orally daily for 14 days. The epithelial defects were drawn and photographed on alternate days, after fluorescein staining. At conclusion, extracts of the corneas were evaluated for collagenase activity. At 14 days, the mean percentage of epithelial defects results in groups 1-3 were 50.0, 50.7, and 7.1%, respectively. Using the Wilcoxon rank sum test (two tailed), the differences were found to be statistically significant (p = 0.0015). Preliminary data indicated that oral doxycycline administration also decreased the collagenase activity in corneas obtained from these animals. Our preliminary findings indicated that systematically administered doxycycline hyclate, 5 mg/kg/day, promotes corneal reepithelialization in the rabbit alkali-burn model, a result, perhaps, of the drug's ability to inhibit excessive collagenase activity.

Administration, Oral↗
PubMed1993

Epithelial basement membrane in alkali-burned corneas in rats. Immunohistochemical study.

We investigated the altered immunolocalization of the components of epithelial basement membrane (BM) in rats with alkali burns of the cornea. Type IV collagen, laminin, and fibronectin were immunohistochemically stained in the alkali-burned corneas after various intervals. Linear laminin immunoreactivity, which represented the localization of the BM, was interrupted, probably by epithelial enzymes, under the regenerating epithelium after 24 h. A normal configuration was restored on day 7, when the central corneal BM showed a type IV collagen immunoreactivity that was not detected in BM of normal cornea. The exact cause of the development of type IV collagen immunoreactivity in the healing BM was not determined. Laminin, type IV collagen, and fibronectin, which were not detected in normal stroma, were all detected in the healing corneal stroma with repopulated keratocytes. Moreover, the BM zone under the regenerating epithelium showed fibronectin immunoreactivity on days 5-14. Distribution of these components of BM seemed to be normal 2 months postburn. The alteration of BM components is thought to be an important marker of the healing process in corneas burned with alkali.

Animals↗
PubMed1994

Suppression of corneal allograft rejection by systemic cyclosporine-A in heavily vascularized rabbit corneas following alkali burns.

Immunologic rejection is the main cause of corneal graft failure, especially in vascularized corneal beds. The purpose of this study was to investigate the effect of systemic Cyclosporine-A (CsA) on the survival of corneal allografts in heavily vascularized rabbit corneal beds, following alkali burn. Heavy corneal vascularization was induced in one eye of 20 rabbits by alkali burn. Forty-five days later, penetrating keratoplasty was performed in all the heavily vascularized corneas. Twenty-five mg/kg/day of CsA was intramuscularly administered to 10 rabbits for 30 days. The other 10 rabbits were treated with the solvent without CsA and were used as a matched control group. The results show a significant difference in corneal allograft survival between the two groups. All corneal grafts in the untreated group were intensely rejected and vascularized within 3 weeks. Nine of the 10 corneal transplants, in the CsA-treated group, remained transparent without signs of immunologic rejection for > 180 days. In one corneal transplant, minor signs of rejection occurred. We suggest that CsA, when given systemically, is a potent drug in the prevention of immunologic rejection in high-risk corneal transplantations, such as allografts, in heavily vascularized corneas following alkali burn.

Animals↗
PubMed1998

Topical use of zinc desferrioxamine for corneal alkali injury in a rabbit model.

PURPOSE: To evaluate the efficacy of topical zinc desferrioxamine in acute corneal alkali injury in rabbits. METHODS: Twenty rabbits were anesthetized and a standardized alkali burn (1N NaOH) was performed in the center of the cornea (7.5-mm diameter). The animals were randomly divided into two groups and treated (double-masked) with topical zinc desferrioxamine, 220 microM, (group 1) or its vehicle (group 2). Drops were applied 7 times/day for 28 days. Topical gentamicin, 0.3%, was instilled twice a day. Animals were evaluated twice a week. At each examination (using the slit-lamp), the depth of corneal ulcer was graded as follows: 0, no ulcer; 1, tissue loss less than one third of corneal thickness; 2, one third to two thirds tissue loss; 3, more than two thirds tissue loss; 4, descemetocele; or 5, perforation. Ulceration area, vascularization, and epithelial defects also were measured. RESULTS: During the study period, the grading of mean corneal ulcerations in group 1 ranged from 0.2 to 1.00, whereas in group 2, it ranged from 1.4 to 2.7. The mean grade and area of ulceration in group 2 were greater than those in group 1 (p < 0.05). CONCLUSION: Topical zinc desferrioxamine may be an adjunctive treatment in protecting the cornea against induced alkali injury.

Administration, Topical↗
PubMed1998

Acute calcification in alkali-injured rabbit cornea treated with synthetic inhibitor of metalloproteinases (SIMP).

PURPOSE: The aim of this study was to evaluate the ultrastructure of acute calcification observed in alkali-injured rabbit corneas treated with synthetic inhibitor of metalloproteinases (SIMP). METHODS: A 12-mm alkali burn was made on the right corneas of 16 rabbits. Eight eyes were treated with topical phosphate-buffered SIMP, and eight control eyes were given the buffer only. Six corneas (five in the SIMP group and one in the control group) developed chalky white opacification in the central area of the cornea within 6-30 days. The corneas were excised for the following examinations: (a) light microscopy, (b) energy-dispersive x-ray analysis, (c) electron microscopy, and (d) high-angle x-ray diffraction. RESULTS: The results confirmed that there was stromal calcification in these corneas, as shown by positive van Kossa staining, needle-shaped calcium crystal aggregates, and increased calcium in the opaque areas of the corneas. Proteoglycans in the corneal stroma of both the SIMP group and the control group significantly decreased after alkali burns. Very few proteoglycan filaments could be identified in the calcified areas of the stroma. X-ray diffraction reflections with a Bragg spacing of 3.44 +/- 0.04 A were observed from all the calcified corneas treated with SIMP. The intermolecular spacings of collagen fibrils in the calcified stroma (1.24 +/- 0.34 nm) were significantly lower than those in the noncalcified stroma (1.64 +/- 0.04 nm) and the control corneas (1.64 +/- 0.02 nm). The results indicate that the collagen molecules within the fibrils of the calcified stroma are packed more closely than normal. CONCLUSION: The comparison of the x-ray diffraction patterns from the calcified corneas and chicken bone suggests that the calcium deposits in these corneas are in a form similar to those found in bone. The higher rate and more extensive calcification in the SIMP-treated corneas may result from the inhibitory action of SIMP on debris processing, but the mechanism is unknown.

Acute Disease↗
PubMed2000

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea.

PURPOSE: We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. RESULTS: The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4. CONCLUSIONS: A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

Animals↗
PubMed2002

ac conductivity spectra of alkali tellurite glasses: composition-dependent deviations from the Summerfield scaling.

We report, for the first time, deviations from the Summerfield scaling in the ac conductivity spectra of single ion conducting glasses. In contrast to the extensively studied borate and germanate glasses, the conductivity isotherms of alkali tellurite glasses do not superimpose upon application of the Summerfield scaling. The deviations depend on the alkali oxide content as well as on the type of the alkali ion. Remarkably, our experimental findings differ considerably from theoretical results describing the hopping dynamics of charge carriers in random barrier landscapes.

Journal Article↗
PubMed2003

Mixed alkali effect in glasses.

We have applied the bond-valence technique to reverse Monte Carlo produced structural models of mixed alkali phosphate glasses in order to elucidate the mixed alkali effect (MAE) in glasses. For the first time, the MAE is reproduced and understood directly from structural models in quantitative agreement with available experimental results. The two types of alkali ions are randomly mixed and have distinctly different conduction pathways of low dimensionality. This implies that A ions tend to block the pathways for the B ions and vice versa, and this is the main reason for the MAE.

Journal Article↗
PubMed2004

Formation and stability of high-spin alkali clusters.

Helium nanodroplet isolation has been applied to agglomerate alkali clusters at temperatures of 380 mK. The very weak binding to the surface of the droplets allows a selection of only weakly bound, high-spin states. Here we show that larger clusters of alkali atoms in high-spin states can be formed. The lack of strong bonds from pairing electrons makes these systems nonmetallic, van der Waals-like complexes of metal atoms. We find that sodium and potassium readily form such clusters containing up to 25 atoms. In contrast, this process is suppressed for rubidium and cesium. Apparently, for these heavy alkalis, larger high-spin aggregates are not stable and depolarize spontaneously upon cluster formation.

Journal Article↗
PubMed2006

Alkali-induced enhancement of surface electronic polarizibility.

From results of ab initio electronic structure calculations based on density functional theory for a set of prototype systems, we find alkali adsorbates to cause a dramatic enhancement of the electronic polarizability of the metal surface extending it several angstroms into the vacuum. This phenomenon is traceable to an unusual feature induced in the surface potential on alkali adsorption. The effect appears to be general, as we find it to be present on metals as varied as Pd and Cu, and helps explain the observed substantial decrease in the vibrational frequency of molecules when coadsorbed with alkalis on metal surfaces. Specifically, for two dissimilar molecules CO and O(2), we trace the softening of the frequencies of their stretching mode when coadsorbed with K on Pd(111) to the enhanced electronic polarizability.

Journal Article↗