The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

On the occurrence of Lymnaea auricularia (Gastropoda: Lymnaeidae) in New Zealand

Abstract

The occurrence in New Zealand of Lymnaea auricularia (L.) is recorded for the first time, and its status world-wide is briefly discussed, with particular reference to its role in economic parasitology. The lymnaeid fauna of New Zealand is briefly reviewed.     

Histological Techniques to Improve Testing of Pulpal Response of Teeth to Filling Material

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pu1p:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation. processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.     

Histological Techniques to Improve Testing of Pulpal Response of Teeth to Filling Material

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pu1p:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation. processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.     

冷光美白技术漂白活髓着色牙的临床疗效分析

目的：评价应用Beyond冷光美白技术漂白活髓着色牙的临床疗效。方法：选择牙齿变色病人117例,其中外源性染色牙18例,氟牙症16例,四环素染色牙47例,增龄性变色牙36例,该组病例的前牙及第一前磨牙均为活髓牙,采用Beyond冷光漂白法治疗,关白效果用Vita比色板进行前后比较。结果：外源性着色牙与增龄性变色牙关白疗效较好,平均提高6．09个色阶。对氟牙症疗效也较明显,但颜色分布不均匀,仍会有条纹或斑块状白斑。对轻、中度的四环素牙平均可提高3．46个色阶,重度四环素牙疗效欠佳。结论：Beyond冷光美白技术是目前治疗活髓变色牙特别简捷、安全和有效的方法之一。     

The biosynthetic routes for carbon monoxide and cyanide in the Ni-Fe active site of hydrogenases are different

The incorporation of carbon into the carbon monoxide and cyanide ligands of [NiFe]-hydrogenases has been investigated by using (13)C labelling in infrared studies of the Allochromatium vinosum enzyme and by (14)C labelling experiments with overproduced Hyp proteins from Escherichia coli. The results suggest that the biosynthetic routes of the carbon monoxide and cyanide ligands in [NiFe]-hydrogenases are different.     

Chelation of divalent cations by lomofungin: role in inhibition of nucleic acid synthesis

Lomofungin inhibition of yeast growth and RNA synthesis is prevented by Cu⁺⁺ or Zn⁺⁺ ions which chelate with the antibiotic and prevent its uptake by the cells. EDTA potentiates the inhibition. Mg⁺⁺ ions do not protect $\text{in vivo}$ or against the inhibition of purified bacterial RNA and DNA polymerases. Lomofungin prevents formation of the RNA polymerase. DNA initiation complex, probably by chelation with the firmly bound Zn⁺⁺ of the enzyme.     

Energetics of SecA dimerization

Transport of many proteins to extracytoplasmic locations occurs via the general secretion (Sec) pathway. In Escherichia coli, this pathway is composed of the SecYEG protein-conducting channel and the SecA ATPase. SecA plays a central role in binding the signal peptide region of preproteins, directing preproteins to membrane-bound SecYEG and promoting translocation coupled with ATP hydrolysis. Although it is well established that SecA is crucial for preprotein transport and thus cell viability, its oligomeric state during different stages of transport remains ill defined. We have characterized the energetics of SecA dimerization as a function of salt concentration and temperature and defined the linkage of SecA dimerization and signal peptide binding using analytical ultracentrifugation. The use of a new fluorescence detector permitted an analysis of SecA dimerization down to concentrations as low as 50 nM. The dimer dissociation constants are strongly dependent on salt. Linkage analysis indicates that SecA dimerization is coupled to the release of about five ions, demonstrating that electrostatic interactions play an important role in stabilizing the SecA dimer interface. Binding of signal peptide reduces SecA dimerization affinity, such that K_d increases about 9-fold from 0.28 μM in the absence of peptide to 2.68 μM in the presence of peptide. The weakening of the SecA dimer that accompanies signal peptide binding may poise the SecA dimer to dissociate upon binding to SecYEG.     

Mineral metabolism and microstructural defects in primate teeth.

There are numerous structural defects that occur in primate teeth due to variable calcification during certain growth stages. These interruptions are usually areas of hypomineralization in enamel and dentin which are referred to as Striae of Retzius and Hunter Shreger bands in the enamel or Incremental Lines of von Ebner and Contour Lines of Owen in the case of the dentin. The frequency of occurrence of these variations in mineralization can be related to such factors as growth tempo, dietary quality, state of health, and past disease episodes. Another structure that appears in the dentin is a region that fails to calcify and is referred to as Inter-globular Dentin. Our studies have shown that the microstructural quality of different species' dentition varies widely. Samples obtained from certain fre-ranging cercopithcoids show that these species have the least hypomineralizations while man has the most. Other primate species range between these two extremems with the pongids nearer to man in these characteristics, as previously noted by Sognnaes. Additionally, out initial study shows a great deal of diversity between prehistoric human populations in the microstructural quality of their teeth. We offer the tentative explanation that this variation is due to differences in the calcitying properties of the diet and hence a difference in the general state of their health.     

冷光美白技术漂白活髓着色牙的临床疗效分析

目的:评价应用Beyond冷光美白技术漂白活髓着色牙的临床疗效。方法:选择牙齿变色病人117例,其中外源性染色牙18例,氟牙症16例,四环素染色牙47例,增龄性变色牙36例,该组病例的前牙及第一前磨牙均为活髓牙,采用Beyond冷光漂白法治疗,美白效果用Vita比色板进行前后比较。结果:外源性着色牙与增龄性变色牙美白疗效较好,平均提高6.09个色阶。对氟牙症疗效也较明显,但颜色分布不均匀,仍会有条纹或斑块状白斑。对轻、中度的四环素牙平均可提高3.46个色阶,重度四环素牙疗效欠佳。结论:Beyond冷光美白技术是目前治疗活髓变色牙特别简捷、安全和有效的方法之一。     

Interaction of Pseudomonas cytochrome cd1 with the cytoplasmic membrane

Labelling with ferritin-conjugated antibody shows that Pseudomonas cytochrome cd₁ is associated with the inner surface of the cytoplasmic membrane. Cytochrome cd₁ is, however, enriched to the soluble fraction obtained after destruction of Pseudomonas spheroplasts. Comparison of the respiratory nitrite reductase activities, due to this cytochrome, between different cellular fractions and the purified enzyme shows that while the kinetic pattern and the temperature dependence of the activity remain almost the same the molecular activity is enhanced when the enzyme is released from cells.A new assay of respiratory nitrite reductase was developed in this study. The method is based on determination of the stoichiometrical proton consumption accompanying nitrite reduction.     

Effect of chelating agents on bud induction and accumulation of iron and copper by the moss Bartramidula bartramioides

The chelating agents, EDDHA, its iron salt, EDTA, and salicylic acid enhance bud formation in Bartramidula bartramioides (Griff.) Wijk & Marg. Salicylic acid elicits optimal response at 10^–4 M , whereas the other substances do so at 10^–7 M . Increased concentration of ferric citrate and cupric sulphate also stimulate bud induction. The accumulation of Fe³⁺ and Cu²⁺ is facilitated by chelators. The endogenous iron content is maximum at 10^–7 M EDDHA or EDTA, which is also the concentration optimal for bud induction.     

The properties of a rat serum protein labelled by the injection of sodium selenite

Properties of a serum selenoprotein which was labelled after the injection of low levels of ⁷⁵SeO₃^2? to rats on a selenium-adequate diet were investigated. When serum was collected three hours after injection the label was incorporated preferentially into one major serum fraction as shown by polyacrylamide gel electrophoresis. Binding was strong, a demonstrated by the fact that very little label was removed by dialysis against 0.60 M NaCl or 0.50 M β-mercapto-ethanol; however, 80% of the ⁷⁵Se was removed by dialysis against 0.50 M NaOH.Molecular weight studies by sodium dodecyl sulfate polyacrylamide gel elecphoresis gave a subunit size of 49 000 under most conditions; when exposed to high concentrations of the detergent (2%) some ⁷⁵Se was associated with a protein having a molecular weight of 25 000. The native selenoprotein eluted before serum albumin on Sephadex G-150 indicating a molecular weight greater than 67 000. The selenoprotein did not coelute with glutathione peroxidase on DEAE-Sephadex A-50.The prior administration of actinomycin D and cycloheximide resulted in a reduction in incorporation of ²⁷Se into serum by 46% and 70%, respectively, which is consistent with the hypothesis that ⁷⁵Se is going into newly synthesized protein. The isoelectric point was determined to be 5.4; when heparin was present, the pI was lowered to 3.2. Treatment with 2 M NaCl did not dissociate the protein · heparin complex, while exposure to 0.25 M β-mercaptoethanol resulted in the dissociation of 60% of the complex.The fact that selenium is so tigthly associated with one serum protein when administered at levels that would be considered normal under most nutritional conditions suggests an important role for this protein, perhaps in the transport of this essential micronutrient throughout the body.     

Ethylenediaminetetraacetic Acid in Ammonium Hydroxide for Reducing Decalcification Time

Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new de-calcification fluid using concentrated ammonium hydroxide (NH₄OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH₄OH required an average of six days. Light microscopy indicated good retention of cellular detail.