Tris-ethylenediaminetetraacetic Acid Research Articles

Comparative evaluation of chlorhexidine gluconate with alcohol and polyhexamethylene biguanide with Tris-EDTA as antiseptic solutions for pre-operative skin preparation in dogs

Background and Aim: Skin antisepsis plays a crucial role in pre-operative skin preparation, with chlorhexidine gluconate and alcohol being historically the preferred choice. However, concerns have risen regarding the development of bacterial resistance to chlorhexidine. Polyhexamethylene biguanide (PHMB) combined with Tris-ethylenediaminetetraacetic acid (Tris-EDTA) has recently emerged as a skin and wound antiseptic. This study aimed to compare the antibacterial efficacy and local safety of 2% chlorhexidine gluconate with 70% alcohol (CG+Alc) and 0.3% PHMB with 6% Tris and 1.86% EDTA (PHMB+Tris-EDTA) for pre-operative skin preparation in dogs. Materials and Methods: Twenty-four adult dogs underwent aseptic preparation on both sides of their ventral abdomens, with one side receiving CG+Alc and the other side receiving PHMB+Tris-EDTA, assigned randomly. Skin swab samples were collected pre-antisepsis and at 3-, 10-, and 60-min post-antisepsis to quantify bacterial colony-forming units (CFUs). Local skin reactions (erythema and edema) were evaluated after hair clipping, pre-antisepsis, and at 3-, 10-, 30-, and 60-min post-antisepsis. Results: There was no significant difference in bacterial CFU counts between the two antiseptic groups pre-antiseptic. Both solutions significantly reduced CFU counts (p < 0.05) at all post-antisepsis sampling times compared with pre-antisepsis. However, dogs treated with PHMB+Tris-EDTA showed a significantly higher incidence of edema at 10 min (p = 0.02) and 30 min (p = 0.003) and a higher incidence of erythema at 10 min (p = 0.043) post-antisepsis compared with CG+Alc. No skin reactions were observed in either group at 60 min post-antisepsis. Conclusion: CG+Alc and PHMB+Tris-EDTA reduced bacterial counts in pre-operative skin preparation in dogs. However, acute transient skin reactions were observed more frequently following the application of PHMB+Tris-EDTA. Keywords: alcohol, antisepsis, chlorhexidine gluconate, dogs, polyhexamethylene biguanide, skin preparation, tris-ethylenediaminetetraacetic acid.

Comparison of Three Methods to Extract Plasmodium falciparum DNA from Whole Blood and Dried Blood Spots.

This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.

Microbial and epidemiological factors in early detection of esophageal squamous cell carcinoma and precancerous lesions.

Microbial and epidemiological factors in early detection of esophageal squamous cell carcinoma and precancerous lesions.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris–ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

Genotoxic Effect of Various forms of Tobacco on Oral Buccal Mucosa and Nuclear Changes as a biomarker.

Aim:The present study aims to assess the genotoxic effect of various forms of tobacco users on buccal mucosa and nuclear changes as biomarkers.Materials and Methods:The study involves 150 cases, they were divided into three groups (two study groups and one control group). The buccal cytological smears were taken from three groups: Group I – 50 smokers, Group II – 50 nonsmokers (smokeless tobacco), and Group III – 50 control group. The buccal cells were transferred into a test tube containing Tris-ethylenediaminetetraacetic acid buffer (pH = 7) and was centrifuged (Remi® 1500 revolution/min [rpm]). Cell suspensions were transferred to the slides and fixed. The slides were stained using PAP and Feulgen stain. The MN and other nuclear abnormalities were studied and compared.Results:Nonsmokers (smokeless tobacco) had significantly increased frequency of all nuclear anomalies compared to smokers and healthy controls. Binucleation, karyorrhexis, micronuclei (MN), karyolysis, broken egg nuclei, and prominent nucleoli in nonsmokers (smokeless tobacco) and condensed chromatin in smokers were the most frequent anomalies. Binucleation and karyorrhexis were significantly more frequent in nonsmokers (smokeless tobacco) compared to smokers. The other nuclear abnormalities were not statistically significant in smokers and nonsmokers (smokeless tobacco).Conclusion:Numerous studies have stated that MN and other nuclear anomalies were present in higher frequency in smokers and nonsmokers. In our study, we found binucleation and karyorrhexis were statistically significant in nonsmokers (smokeless tobacco) compared to smokers. The other nuclear anomalies showed insignificant results. In order to further validate the significance of this study, a larger sample size has to be studied. On comparing the staining efficacy of smokers and nonsmokers using PAP and Feulgen stain, both the stains showed positive results. In the present study, DNA-specific Feulgen stain shows better staining of nuclear anomalies compared to DNA nonspecific PAP stain, which was found to be statistically significant.

Extraction and Evaluation of Outer Membrane Vesicles from Two Important Gut Microbiota Members, Bacteroides fragilis and Bacteroides thetaiotaomicron.

Objective The gastrointestinal tract (GI) is colonized by a complex microbial community of gut microbiota. Bacteroides spp. have significant roles in gut microbiota and they host interactions by various mechanisms, including outer membrane vesicle (OMVs) production. In the present study, we extracted and assessed Bacteroides fragilis (B. fragilis) and Bacteroides thetaiotaomicron (B. thetaiotaomicron) OMVs in order to evaluate their possible utility for in vivo studies. Materials and Methods In this experimental study, OMVs extraction was performed using multiple centrifugations and tris-ethylenediaminetetraacetic acid (EDTA)-sodium deoxycholate buffers. Morphology, diameter, protein content, profile, and lipopolysaccharide (LPS) concentrations of the OMVs were assessed by scanning electron microscopy (SEM), nanodrop, Bradford assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the Limulus Amoebocyte Lysate (LAL) test, respectively. Zeta potential (ζ-P) was also assessed. The viability effect of OMVs was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in Caco-2 cells. Results Spherical OMVs with diameters of 30-110 nm were produced. The OMVs had different protein profiles. The LPS concentrations of the B. fragilis and B. thetaiotaomicron OMVs were 1.80 and 1.68 EU/mL, respectively. ζ-P of the B. fragilis OMVs was -34.2 mV and, for B. thetaiotaomicron. it was -44.7 mV. The viability of Caco-2 cells treated with OMVs was more than 95%. ConclusionThe endotoxin concentrations of the spherical OMVs from B. fragilis and B. thetaiotaomicron were within the safe limits. Both OMVs had suitable stability in sucrose solution and did not have any cytotoxic effects on human intestinal cells. Based on our results and previous studies, further molecular evaluations can be undertaken to design OMVs as possible agents that promote health properties.

The effect of saturated and unsaturated fatty acids on the production of outer membrane vesicles from Bacteroides fragilis and Bacteroides thetaiotaomicron.

The aim of present study is to investigate the effect of fatty acids on the outer membrane vesicles (OMVs) produced by Bacteroides spp. Bacteroides spp. is the important member of Gut microbiota that employ OMVs production for interact with host. Besides, dietary fatty acids could influence on determination of gut microbiota composition and immune response. In this regard, we evaluated the effect of fatty acids on the growth and OMVs production of Bacteroides fragilis and Bacteroides thetaiotaomicron. B. fragilis and B. thetaiotaomicron were grown on BHI broth with and without palmitic and palmitoleic acids as saturated and unsaturated fatty acids, respectively. OMVs were extracted using multiple centrifugation and tris-ethylene diamine tetra acetic acid (EDTA)-Sodium deoxy cholate buffers. Physicochemical properties of OMVs were detected by electron microscopy (SEM), Bradford Coomassie brilliant blue assay and SDS-PAGE. Data were analyzed with One-way ANOVA using SPSS. The growths of both Bacteroides were significantly increased by palmitic acid. Nevertheless, palmitoleic acid had no significant effect on them. Palmitic acid significantly decreased and increased the production of B. fragilis OMVs at low and high concentration, respectively. However, the production of B. thetaiotaomicron OMVs was not significantly affected by palmitic acid. Although palmitoleic acid had a significant decreasing effect on the production of B. fragilis OMVs, it significantly increased the production of B. thetaiotaomicron OMVs at low concentration. In conclusion we reported that palmitic acid had a stimulatory effect on the growth of B. fragilis and B. thetaiotaomicron and had a dose dependent effect on the production of B. fragilis OMVs. Also producing of B. thetaiotaomicron OMVs was affected by palmitoleic acid in a dose dependent manner.

Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery.

Small interfering RNA (siRNA) delivery is a prospective method in gene therapy, but it has application limitations such as negative charge, water solubility and high molecular weight. In this study, a safe and efficient nano-vector, CRS, was designed and synthesized to facilitate siRNA delivery. Physical and chemical properties of VEGF-siRNA/CRS were characterized by methods including scanning electron microscopy (SEM), transmission electron microscopy, zeta potential (ζ) measurement, drug-releasing rate measurement, gel electrophoresis and confocal microscopy. The biological activities were evaluated using cell viability assay, gene-silencing efficacy assay in vitro, real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA) and antitumor tests in vivo. The mean nanoparticle size of VEGF-siRNA/CRS was 121.4±0.3 nm with positive ζ potential of 7.69±4.47 mV. The release rate of VEGF-siRNA from VEGF-siRNA/CRS was 82.50% sustained for 48 h in Tris-ethylenediaminetetraacetic acid buffer (pH 8.0). Real-time polymerase chain reaction was used to analyze the efficiency of the transfection, and the result showed that VEGF mRNA expression had been knocked down by 82.36%. The expression of VEGF protein was also recorded to be downregulated to 14.83% using ELISA. The results of cytotoxicity measured by Cell Counting Kit-8 assay showed that VEGF-siRNA/CRS had significant inhibitory effect on HeLa cells. The results of antitumor assays indicated that VEGF-siRNA/CRS exhibited tumor cell growth inhibition in vivo. The results demonstrated that VEGF-siRNA could be delivered and transported by the designed carrier, while siRNA could be released constantly and led to an increasing gene-silencing effect against VEGF gene. In conclusion, VEGF-siRNA/CRS is a promising carrier for siRNA delivery, and further studies are warranted.

Quantification and qualification of bacteria trapped in chewed gum.

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including caries and periodontal disease, are caused by bacteria. However, it is unknown whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the first method, known numbers of bacteria were finger-chewed into gum and chewed gums were molded to standard dimensions, sonicated and plated to determine numbers of colony-forming-units incorporated, yielding calibration curves of colony-forming-units retrieved versus finger-chewed in. In a second method, calibration curves were created by finger-chewing known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested to chew gum up to 10 min after which numbers of colony-forming-units and total numbers of bacteria trapped in chewed gum were determined using the above methods. The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a slow decrease over time up to 10 min was observed. Around 108 bacteria were detected per gum piece depending on the method and gum considered. The number of species trapped in chewed gum increased with chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria from the oral cavity.

Experimental study on dissolution of calcium oxalate calculi with Tris-ethylenediaminetetraacetic acid buffer and trypain

Objective To study the safety and efficiency of Tris-ethylenediaminetetraacetic acid (TE) buffer with trypain for dissolution of calcium oxalate calculi in vitro.Methods Ten patients with calcium oxalate crystals stones were collected clinically.Calcium oxalate calculus was dissolved with the litholytic agents ethylenediaminetetraacetic acid (EDTA),TE buffe or TE buffer with trypain.The calcium of irrigating solution was determined by atomic absorption spectrophotomatry.Dry weighing stones before and after weighing,and the surface of irrigated calculi were observed by scanning electron microscope.The pathological changes of bladder mucosae were observed after the rabbit bladders were irrigated.Results The calcium concentration in TE with trypain group [(667.96 ± 46.24) μg/L] was significantly higher than in EDTA group [(414.95 ±50.04) μg/L],TE group [(439.48 ±57.21) μg/L],and EDTA with trypain group [(503.42 ±57.16) μg/L,P ＜0.05].There was no obvious damage under the light microscope.Conclusion TE buffer solution plus trypain dissolved calcium stones with better effect and little damage.It is expected to be further tested and to be applied in the clinical practice for the stone treatment as a new method. Key words: Calcium oxalate calculi; Tris-ethylenediaminetetraacetic acid buffer; Trypain

Measurement of the interaction forces at various pH levels by using AFM for the interpretation of DNA adsorption on silanized surfaces

Various surfaces have been used for deoxyribonucleic acid (DNA) immobilization, one example being a silanized surface. This is useful for determining DNA lengths and, thus, locating specific gene sequences in DNA by using fluorescence microscopy and scanning probe microscopy. In this study, we deposited DNA by using the molecular combing method and, we used fluorescence microscopy to study how the chain lengths of n-alkylsilanes affected the surface density of DNA deposited on the silanized surfaces in a tris-ethylenediaminetetraacetic acid (TE) buffer. The forces between a cleaned silicon-nitride (Si3N4) tip and each substrate surface in aqueous buffers at various pH levels (1.0 ~ 9.0) were also studied by using atomic force microscopy to measure the force-distance curves. We explain why the density of lambda bacteriophage DNA (λ-DNA) deposited by using the molecular combing method at pH 8 was lower on the silanized surface with the shorter alkyl chain than it was on the silanized surface with the longer alkyl chain in terms of the electrical double layer (EDL) and the adhesive force.

Expression Pattern and Subcellular Localization of Human Papillomavirus Minor Capsid Protein L2

The expression pattern of human papillomavirus (HPV) capsid antigen L2 is poorly described, and the significance of its localization with both promyelocytic leukemia protein (PML) and Daxx in a subnuclear domain, nuclear domain 10 (ND-10), when ectopically expressed in tissue culture cells is controversial. To address whether ND-10 localization of L2 occurs in natural cervical lesions, we used a HPV16 and HPV18 L2-specific monoclonal antibody (RG-1), in addition to rabbit antiserum to HPV6 L2, to localize L2. Immunohistochemical staining with RG-1 produced diffuse staining in the nuclei of some cells located within the superficial epithelial layers in eight of nine cases of HPV16/18(+) cervical intraepithelial neoplasia grade 1 (CIN1); however, no staining was observed in HPV16/18(+) high-grade CIN (0 of 8 cases), normal cervical epithelium (0 of 20 cases), cervical squamous cell carcinoma (0 of 102 cases), adenocarcinoma (0 of 51 cases), or adenosquamous carcinoma (0 of 6 cases). HPV16/18(+) cervical lesions that express L2 exhibit higher HPV16/18 genome copies per cell compared with those that do not positively stain with RG-1 (P = 0.04). RG-1 staining of HeLa cells transfected with L2 expression constructs was frequently concentrated in the ND-10, particularly in cells expressing high levels of L2, and co-localized with the cellular markers of ND-10, PML, and Daxx. In contrast, L2 was primarily diffuse within the nucleus and distinct from ND-10 as defined by PML immunofluorescent staining in CIN lesions, condylomata, and HPV16-transduced organotypic cultures.

Ability of Reptalus quinquecostatus (Hemiptera: Cixiidae) to inoculate stolbur phytoplasma to artificial feeding medium

AbstractLaboratory trials were carried out on wild individuals of Reptalus quinquecostatus (Cixiidae), a potential vector of stolbur phytoplasma to grapevine, to assess its ability to inoculate the phytoplasma in artificial feeding medium. Seventy‐seven specimens of the cixiid were tested on a sucrose–TE (Tris–ethylenediaminetetraacetic acid) diet and 62 of them survived less than 24 h. Polymerase chain reaction (PCR) assays performed on the insect bodies detected the presence of stolbur phytoplasma, with an infection rate of 32.5%. Restriction fragment length polymorphism analysis of the tuf gene, amplified by PCR, revealed Vergilbungskrankheit type I (VK‐I) in 20 specimens, VK‐II in 4 specimens and both types in 1 specimen. Ten of the 25 infected R. quinquecostatus specimens successfully inoculated VK‐I in the sucrose solution, that is, a 40% inoculation efficiency despite the brief survival. The results indicate that R. quinquecostatus is a competent species to transmit the stolbur phytoplasma in artificial conditions. The repeated observation of adults feeding on grapevine strengthens the hypothesis that the species is a vector of stolbur phytoplasma to this plant.

High‐throughput Procedure for Single Pollen Grain Collection and Polymerase Chain Reaction in Plants

Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.

Epigenetic Inhibition of Nuclear Receptor Small Heterodimer Partner Is Associated With and Regulates Hepatocellular Carcinoma Growth

Aberrant hypermethylation of promoter regions in cytosine-guanine dinucleotides (CpG) islands has been shown to be associated with transcriptional silencing of tumor-suppressor genes in many cancers. This study evaluated the methylation profile and the tumor-suppressive function of the small heterodimer partner (SHP, NR0B2) in the development of human hepatocellular carcinoma (HCC). Human HCC pathologic specimens and cell lines were used as model systems in this study. The expression of SHP is diminished in HCC pathologic specimens and cell lines by epigenetic silencing owing to SHP promoter hypermethylation. In vitro methylation decreased SHP promoter transactivation and nuclear receptor LRH-1 binding, an event that was reversed by demethylation. Overexpression of SHP inhibited HCC foci formation, arrested HCC tumor growth in xenografted nude mice, and increased the sensitivity of HCC cells to apoptotic stimuli. Further analysis of a total of 19 normal liver and 57 HCC specimens showed that down-regulation of SHP gene expression may be a common denominator of HCC. We propose that SHP functions as a novel tumor suppressor in the development of HCC. These findings provide new insight into the molecular mechanisms leading to this common cancer and may have both diagnostic and therapeutic applications.

Suitability of DNA Extracts from Peripheral Blood, Oral Mucosa, Dental Pulp, and Split Tooth for Genetic Analysis

Objective: To assess the suitability of DNA extracted from peripheral blood, oral mucosal tissue, dental pulp, and split tooth for genetic analysis.Patients and Methods: DNA was isolated and extracted from the blood, oral mucosal tissue, dental pulp, and split tooth of 5 patients undergoing surgical removal of an impacted third molar. Six millilitres of peripheral blood was collected in an ethylenediamine tetra-acetic acid tube and frozen immediately. A piece of tissue from the buccal mucosal flap was removed following surgery. The pulp was harvested by cutting the extracted molar in half and scraping with a curette. DNA was extracted from both halves of the tooth. All samples were extracted in Tris-ethylenediamine tetra-acetic acid (pH 8) using sodium dodecyl sulphate and proteinase K, and digested overnight at 37°C. DNA was purified using phenol-chloroform-isoamyl alcohol, precipitated in 4 M sodium acetate with ethanol, and assessed by amplification of the variable number of tandem repeat locus DIS80 using polymerase chain reaction. The presence of contaminating bacteria in the samples was analysed by detection of the bacterial 16S ribosomal gene.Results: The DNA purity in 90% of the samples was 1.5 to 1.7, which indicated good purity with minimal protein contamination. Amplification of the extracted DNA at the variable number of tandem repeat locus DIS80 produced distinct bands in DNA extracted from the 4 different samples. Individuals homozygous for the DIS80 locus in chromosome 1 produced equal numbers of repeats from both homologues of chromosome 1. DNA from heterozygous individuals produced 2 bands as a result of amplification of a different number of repeats in the 2 chromosomes.Conclusion: This preliminary study indicates that pure and amplifiable DNA can be successfully extracted from blood, oral mucosal tissue, dental pulp, and split tooth.

A Dielectric Biosensor Using the Capacitance Change with AC Frequency Integrated on Glass Substrates

Glass-based microchannel chips were fabricated using photolithographic technology, and Pt thin-film microelectrodes as dielectric biosensors were integrated on them. From capacitance-frequency measurements at various interelectrode distances and ionic concentrations, a significant difference between deionized (DI) water and tris-ethylenediaminetetraacetic acid (EDTA) (TE) buffer was observed in the low-frequency region. Although the capacitance (CM) of the DI water decreased as the interelectrode distance increased, that of the TE buffer was similar up to a frequency of 100 Hz, after which it was spilt in the same manner as the DI water above 100 Hz. As the ionic concentration increased, the CM of the TE buffer increased and the slope in the low frequency region changed from -0.875 to -0.425. The point where the slope changed shifted towards the frequency increase. These observations were clarified from the viewpoint of interfacial phenomena, such as the electrical double layer and Faradaic reactions, the dielectric constant related to conductivity, and the capacitance inversely proportional to the interelectrode distance. The addition of deoxyribonucleic acid (DNA) molecules (10 ng/µl) increased the capacitance and dielectric loss in the TE buffer at low frequency. It is feasible to use dielectric properties for the rapid and direct detection of biomolecules, particularly DNA molecules, without using labels or indicators.

The Effect of Lyophilization on Plasmid DNA Activity

The effect of lyophilization of plasmid DNA's ability to express an encoded protein was studied. Plasmid DNA, pRL-CMV expressing Renilla luciferase, was purified and stored in Tris-ethylenediaminetetraacetic acid (EDTA) buffer. Aliquots of the plasmid were lyophilized using analytical equipment, both alone and in the presence of carbohydrate. Samples were rehydrated and subject to functional and structural analyses. Analytical techniques included transfection efficiency in COS-1 cells, agarose gel electrophoresis, dimethylethylenediamine (DMED) assay for abasic sites, circular dichroism measurement, and UV spectroscopy. The lyophilization of pRL-CMV plasmid DNA resulted in a statistically significant loss of transfection efficiency (p < 0.05). Mono- and disaccharides could completely restore transfection efficiency. Agarose gel electrophoresis and the DMED assay demonstrated no change in gross plasmid structure or increase in abasic sites during lyophilization, respectively. Changes in DNA form, as measured by a change in ellipsisity, were observed on lyophilization. However, these changes were transient and were not shown to be responsible for loss of transfection efficiency. A hyperchromic effect was observed at 260 nm after lyophilization and could be reversed by the presence of carbohydrates. Lyophilization causes a decrease in plasmid DNA activity as measured by an in vitro transfection assay. Carbohydrates can ameliorate this decreased activity, which may be due to structural changes seen during the lyophilization process.

Cell envelopes of gram negative bacteria: composition, response to chelating agents and susceptibility of whole cells to antibacterial agents.

The effects of ethylenediamine tetraacetic acid (EDTA) and related chelating agents on the sensitivity of isolated cell envelopes of some β‐lactamase +ve and ‐ve strains of Gram negative bacteria have been investigated. Envelopes from Pseudomonas aeruginosa (especially strain NCTC 1999) contained the greatest amounts of Mg2+ and were the most sensitive to these agents in terms of (i) lysis, (ii) release of cations, (iii) release of readily extractable lipid. Cyclohexane—1,2, ‐diamine‐tetraacetic acid was the most effective chelator, followed by EDTA and N‐hydroxy‐ethylethylenediamine triacetic acid, with nitriloacetic acid and iminodiacetic acid having little effect. A lysozyme–Tris–EDTA system also caused lysis of P. aeruginosa envelopes. The sensitivity of whole cells of the various strains to some β‐lactam antibiotics and other antibacterial agents has been carried out and the basis of sensitivity or resistance in relation to drug destruction and the above envelope composition discussed.

Haemoglobin polymorphism in the orangutan and an animal with four major haemoglobins.

IN examining haemolysates from ten orang utans from the Yerkes Regional Primate Center collection we found four different haemoglobin phenotypes including one in which three major haemoglobin zones were present. The samples, kindly supplied by Prof. G. Bourne and Dr. J. Moor-Jankowski, were first analysed on starch gels using a tris-ethylenediamine tetraacetic acid (EDTA) buffer system1 and were later investigated by hybridization2,3 of the component haemoglobins with various other haemoglobins.