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PubMed1998

The sodium hydroxide erosion assay: a revision of the alkali resistance test.

Burckhardt proposed the alkali resistance method as a means of assessing the integrity of the stratum corneum barrier in 1947. Researchers after Burckhardt largely found the test unreliable and nonreproducible; it therefore fell into disuse worldwide. We have upgraded the procedure by exposing the skin to 1.0 M sodium hydroxide under strictly specified conditions for successive 1-min periods until the emergence of the first erosions, revealed by staining with nitrazine yellow. Histology showed that the erosions were follicular and limited to the epidermis. The test was highly reproducible and repeatable. We demonstrated the usefulness of the test in the following ways: (1) the erosion time increased with aging, correlating with a thickened horny layer; (2) as few as five Scotch tape strippings greatly decreased the erosion time, although transepidermal water loss was only slightly increased; (3) slight damage to the horny layer by a 24-h exposure to 0.01% sodium lauryl sulfate sharply reduced the erosion time; (4) the erosion time decreased after daily open applications for 3 weeks of clobetasol propionate, corresponding to the thinned horny layer; (5) daily applications of petrolatum increased the erosion time. This new version of the alkali resistance test, renamed the sodium hydroxide erosion assay, promises to be a useful tool for studying the horny layer barrier in healthy and diseased skin.

Administration, Topical↗
PubMedDate not supplied

Effects of the matrix metalloproteinase inhibitor GM6001 on the destruction and alteration of epithelial basement membrane during the healing of post-alkali burn in rabbit cornea.

PURPOSE: To examine the alteration in structure and matrix composition of epithelial basement membrane (BM) during the healing of alkali-burned rabbit cornea, and the roles of matrix metalloproteinases (MMPs) in these alterations. METHODS: The central cornea of one eye of 78 albino rabbits was exposed to 1 N NaOH for 180 s under general and topical anesthesia and allowed to heal with or without subconjunctival injection of GM6001 (an MMP inhibitor). Cryosections of affected corneas were observed by H&E staining, immunohistochemistry for type IV collagen subtypes, or in situ zymography for detection of localization of MMP activity. RESULTS: Uninjured corneal epithelial BM exhibited alpha5 (IV)-immunoreactivity, but lacked the alpha1/alpha2-immunoreactivity of collagen IV. Epithelial BM in healing burned cornea transiently exhibited alpha1/alpha2-immunoreactivity. Examination by in situ zymography showed an upregulation of MMP activity in the regenerated central epithelium and anterior stroma of the burned corneas at days 7 and 14. GM6001 suppressed degradation of alpha5-containing epithelial BM in vivo and also in organ culture. CONCLUSIONS: Epithelial BM was degraded by endogenous MMPs during healing following an alkali burn in rabbit cornea. GM6001 had an inhibitory effect on the degradation of the epithelial basement membrane in burned cornea in vivo.

Animals↗
PubMed1982

Chronic acidosis with metabolic bone disease. Effect of alkali on bone morphology and vitamin D metabolism.

Chronic metabolic acidosis and osteomalacia developed in two patients following urinary diversion. Good clinical, biochemical, and histologic responses were seen following treatment with alkali alone (vitamin D was not given), despite the presence of markedly impaired glomerular filtration in one of the patients. Plasma 25-hydroxyvitamin D and 1 alpha, 25-dihydroxyvitamin D concentrations were normal before and during treatment in one of the patients and in the other were low before and normal during treatment. The results show that successful treatment of the osteomalacia of chronic acidosis is not necessarily accompanied by changes in the plasma levels of vitamin D metabolites and that even when marked glomerular dysfunction coexists with acidosis and osteomalacia, treatment with alkali may be more appropriate than the administration of vitamin D analogues.

Acidosis↗
PubMed1990

A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation.

Alkali-stable phosphorylation of proteins, particularly phosphotyrosine and phosphohistidine, is an important phenomenon in cells. In the case of phosphohistidine and some other phosphoamino acids, the phosphorylation is acid-labile and in these cases studies have been severely limited by the absence of a rapid assay suitable for acid-labile phosphorylation. The assay presented here involves a conventional kinase assay reaction followed by mild alkaline hydrolysis and adsorption of the product to washed Nytran paper at high pH. After further washing, at pH 9, the radioactivity on the papers is determined by liquid scintillation counting. Hence, acid-labile phosphorylation is preserved. The assay is selective for alkali-stable phosphorylation but not fully specific, mainly due to the need to balance the severity of the partial alkaline hydrolysis with the stability of the protein-peptide bonds. The assay has been used for the purification and characterization of a protein histidine kinase from Saccharomyces cerevisiae.

Histidine↗
PubMed1975

Alkali-induced reduction of the beta-cytochromes in purified complex III from beef heart mitochondria.

Approx. 40-50% of the cytochrome b in purified Complex III is reduced by ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine or phenazine methosulfate at neutral pH. The remaining cytochrome b, including cytochrome b-565, is reduced by increasing the pH. The apparent pK for this reduction is between pH 10 and 11, and is more than two pH units higher than a similar alkali-induced transition in Mg-ATP particles. Alkali-induced reduction of cytochrome b occurs concomitantly with the exposure of hydrophobic tyrosine and tryptophan residues to a more hydrophilic environment. The relationship of these findings to the presence of a substrate accessibility barrier in Complex III is discussed.

Adenosine Triphosphate↗
PubMed1976

Effects of alkali metal cations on phospho-enzyme levels and [3H] ouabain binding to (Na+ + K+)-ATPase.

The effects of several alkali metal cations on the relationship between steady state phospho-enzyme levels and initial velocity and equilibrium levels of [3H]-ouabain binding to (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3.) were examined. Only Na+ increased both phospho-enzyme and [3H] ouabain binding levels above those observed in the presence of Mg2+ alone. While Na+ stimulated phosphorylation with an apparent Km of about 1 mM, its stimulation of [3H] ouabain binding was biphasic, the lower Km for stimulation corresponding to the Km for formation of phospho-enzyme. Among the other alkali metal cations, potassium, rubidium and lithium were at least eight times more effect in reducing phospho-enzyme levels than in reducing [3H] ouabain binding. This discrepancy is not due to the stability of the enzyme-ouabain complex, nor to any action on the rates of formation or dissociation of the enzyme-ouabain complex. The data thus suggest that [3H] ouabain interacts with the K+, Rb+ or Li+ -enzyme complexes. For Li+, this hypothesis is further supported by the observation that Li+ can cirectly increase the equilibrium level of [3H] ouabain binding to this enzyme under certain conditions.

Adenosine Triphosphatases↗
PubMed1985

Alkali lability and rapid initiation of excision repair following photoaffinity damage by ethidium azide.

DNA damage and repair provoked by ethidium azide (EA) photoaffinity labeling in mouse leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on ice, but restoration of DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated viral DNA with EA failed to introduce DNA strand breaks. However, it was discovered that photoaffinity labeling of DNA with EA resulted in alkali labile sites shown by single strand breaks produced on alkaline sucrose sedimentation or by alkali exposure followed by sedimentation on neutral formamide gradients. These results suggest that the drug attachment sites should be identifiable by the location of such single strand breaks.

Affinity Labels↗
PubMed1987

A method to exchange alkali light chains on myosin subfragment 1.

Exchange of bound alkali light chains on myosin by free alkali light chains is described. It was found that the yield of hybrid obtained was dependent on the incubation time in 4.7 M NH4Cl at pH 9.5. 60% recovery of S1(A1) from S1(A2) was obtained using only a 2-fold molar excess of A1 over S1(A2).

Actins↗
PubMed1985

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy.

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).

Animals↗
PubMed1975

A sublingual-submandibular calcific mass associated with tumoral calcinosis in a patient with suspected milk-alkali syndrome.

The milk-alkali syndrome is a disorder with diverse clinical and biochemical manifestations. It includes three known clinical syndromes. A case of milk-alkali syndrome with associated tumoral calcinosis is presented. The patient developed an unusual oral mass as part of the disease complex. Treatment consisted of excision of the intraoral mass, diet management, and surgical correction of the associated ulcer disease.

Aged↗
PubMed1986

Sequence-specific alkali-labile lesions in DNA caused by D-isoglucosamine.

The site-specific induction of DNA damage by 1-amino-1-deoxy-D-fructose (D-isoglucosamine) was investigated. When 32P-end-labeled DNA restriction fragments of known sequence were reacted with D-isoglucosamine in the presence of Cu2+, and the DNA products were analyzed on high-resolution denaturing polyacrylamide gels after treatment with aqueous piperidine (1 M) at 90 degrees C for 30 min, the DNA strands were cleaved at pyrimidine residues at a statistically significant frequency, and 80.5% of the extensively damaged sites were induced at pyrimidine residues in dinucleotide sequences of pyrimidine-purine (5'----3'). These cleavages were scarcely observed without piperidine/heat treatment. The damaged DNA sites increased in proportion to the reaction time and concentration of D-isoglucosamine. Metal-chelating agents (EDTA, diethylenetriaminepentaacetic acid) and some oxygen radical scavengers inhibited the induction of alkali-labile lesions. These results indicate that some oxygen radicals are involved in the induction of alkali-labile lesions.

Base Sequence↗
PubMed1996

Yersinia spp. and numbers, with particular reference to Y. enterocolitica bio/serotypes, occurring on Irish meat and meat products, and the influence of alkali treatment on their isolation.

Samples (n = 340) of Irish meat and meat products were examined to determine the incidence and initial numbers of Yersinia spp. present. In cases where no typical Yersinia colonies were observed, samples were cold enriched up to 21 days at 4 degrees C and streaked out on CIN agar with and without post-enrichment alkali treatment. The highest isolation rates of Yersinia were found using post-enrichment alkali treatment and an incubation temperature of 37 degrees C. All Yersinia isolates were identified to species level and Y. enterocolitica isolates were biotyped and serotyped. Pathogenic isolates (biotypes 1B-5) were examined for the presence of a virulence plasmid using dye binding reactions and agglutination tests. The incidence of Yersinia spp. was high on raw (89%) and cooked meats (49%) obtained fromdelicatessen counters but lower in pre-packaged cooked meats (18.8%). Initial numbers of Yersinia were generally < 2.7 log10cfu/g on raw meats and < 0.7 log10cfu/g on cooked.

Food Microbiology↗
PubMed1978

Activity coefficients of salts in highly concentrated protein solutions. I. Alkali chlorides in isoionic bovine serum albumin solutions.

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KCl, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt%, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behavior of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl- ion binding to BSA and BSA bound "non-solvent" water with probably electrostatic long range interactions of the BSA(Cl-)nu polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed +/- 2%.

Biological Transport↗
PubMed1983

Alkali-extraction of membranes from mouse erythrocytes infected with Plasmodium berghei.

Alkali-extracted membrane material from hypotonically lysed Plasmodium berghei-infected mouse erythrocytes has been analyzed by dodecyl sulphate polyacrylamide gel electrophoresis and fractionated by discontinuous sucrose gradient centrifugation. Components characteristic for the protein pattern associated with P. berghei infection have been recovered in the alkali-extracted material. Parasite components, free of host cell membrane contamination, have been obtained as a high-density fraction from sucrose gradients. This fraction is characterized by polypeptides with apparent molecular weight of 71,000 and 52,000.

Animals↗
PubMed1990

Possible presence of the difference peptide in alkali light chain 1 of fish fast skeletal myosin.

1. Presence of N-terminal peptide ("difference peptide") in alkali light chain 1 (A1) of fish fast skeletal myosin was examined by comparing two kinds of light chain-based myosin subfragment 1 (S1) isozymes from the yellowtail Seriola quinqueradiata. 2. On tryptic digestion, A1 was cleaved to a smaller fragment (mol. wt decrement by 2000) along with the cleavage of S1 heavy chain, while A2 was resistant to trypsin. Two-dimensional gel electrophoresis showed that A1 released a basic peptide by tryptic digestion. 3. Both S1 isozymes showed clear kinetic differences in actin-activated Mg-ATPase activity, suggesting a higher affinity of A1 for actin. Affinity of A2 for heavy chain was also estimated to be about 2-fold higher than that of A1, as judged by the model experiments in which rabbit S1 isozymes were hybridized with heterologous alkali light chains.

Actins↗
PubMed1995

Effect of EGF dosage forms on alkali burned corneal wound healing of mice.

The local treatment effects of EGF forms on alkali burned mice corneal wounds were identified. The corneal wounds were induced by 0.5 M NaOH solution on the corneal surfaces of the mice. The local epidermal growth factor solutions (100 ng/ml) and gel form in 0.2 per cent w/w carbopol 940 (100 ng/ml) were dropped in 5 microliters aliquots into the eye twice a day. The corneal wounds were measured for 15 days at 7-day intervals and examined histologically at the end of 15th day of the experimental period. The results indicated that topical epidermal growth factor treatment in solution improved the healing of alkali burned corneal wounds when compared with epidermal growth factor delivered in a polymer system.

Administration, Topical↗
PubMed1989

The tissue specificity of the age-related changes in alkali-induced DNA-unwinding.

To determine if the age-related changes in chromatin digestibility are tissue-specific, fluorometric analysis of the alkali-induced DNA unwinding technique was adapted for soft-tissue chromatin studies. The rate of DNA unwinding in the brain and liver of young Fischer 344 male rats (3 months of age) was significantly greater than the rates measured in middle-aged (15 months) or aged rats (26 months). In contrast, the rate of DNA unwinding in the intestinal epithelium, a continuously replicating tissue, did not significantly vary with age. Although this assay is capable of detecting DNA strand breaks in vivo following N-nitrosodimethylamine administration, the age-related changes could not be attributed to reduced DNA strand lesions in the aged animals. The % double-stranded DNA at time 0 of incubation in alkali was lower in the brain and liver of aged rats indicating that DNA strand breaks may actually increase with aging. These results indicate that proliferative activity of the tissue is an important determinant of age-related changes in chromatin structure.

Aging↗
PubMed2006

Use of topical human amniotic fluid in the treatment of acute ocular alkali injuries in mice.

PURPOSE: To evaluate the efficacy of topical human amniotic fluid (HAF) in the treatment of ocular acute alkali burns in mice. DESIGN: Experimental study. METHODS: A chemical burn with 2 microl of sodium hydroxide 0.15 mol/l was created in one eye of 30 mice. The animals were divided into gender- and age-matched groups according to the topical treatment that was administered: group 1 was treated with preterm HAF (n = 10 mice); group 2 was treated with term HAF (n = 10 mice), and group 3 was treated with saline solution (n = 10 mice). Treatment consisted of one drop that was applied to the burned eye five times per day (week one), and three times per day (week two). The epithelial defect was photographed and measured on days two and four. Ocular burn damage was assessed at days two, seven, and 14 after a pre-established classification. On day 14, both eyes of each mouse were enucleated and assessed histopathologically. RESULTS: Median epithelial defect (interquartile range [IQR], 25th, 75th percentile) at day four was 9.93% (IQR, 8.57, 11.27) for group 1, 7.30% (IQR, 5.96, 8.97) for group 2, and 18.92% (IQR, 11.71, 27.64) for group 3 (P < .0076). The overall change (difference in slope) in ocular burn score between days 2 and 14 was -0.127 (P = .009) in group 1 vs 3, -0.134 (P = .012) in group 2 vs 3, and 0.007 (P = .88) in group 1 vs 2. On histologic examination saline solution-treated corneas had more inflammatory cells and blood vessels than HAF-treated corneas. CONCLUSION: Topical preterm/term HAF was an effective topical therapy for limiting the damage after acute alkali burns of the eye in this animal model.

Acute Disease↗