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Introducción: Las mioclonías son contracciones musculares paroxísticas de corta duración o pérdida abrupta del tono muscular, denominadas mioclonías positivas y negativas, respectivamente. Se presenta un caso clínico de mioclonías positivas y negativas generalizadas y se pretende describir los múltiples mecanismos fisiopatológicos y etiologías que lo desencadenan. Presentación del caso: Hombre de 35 años, con diabetes mellitus tipo 1 complicada con enfermedad renal diabética en hemodiálisis, desarrolló una bacteriemia asociada a catéter por Staphylococcus aureus y presentó mioclonías positivas y negativas. Se identificaron como posibles desencadenantes la uremia, la infección y los fármacos con potencial promioclónico; el hallazgo incidental de una lesión isquémica en núcleo caudado no explicaba la semiología encontrada en el paciente. Se hizo el control y retiro de todos los factores promioclónicos enunciados, junto a manejo farmacológico con levetiracetam, y con ello se logró el control de los síntomas. Discusión: Los pacientes con enfermedad renal crónica son susceptibles a la acumulación de productos tóxicos de tipo guanidinas, que tienen potencial para producir mioclonías. Además, las infecciones, el uso de fármacos con potencial promioclónico y lesiones estructurales como las isquemias corticales son etiologías que deben considerarse en el diagnóstico diferencial. El mayor impacto en los síntomas se observa con el control del factor desencadenante, y, en caso de persistir, la terapia farmacológica proporciona buenos resultados. Conclusión: Las mioclonías son trastornos del movimiento relativamente comunes en la enfermedad renal crónica. La identificación del desencadenante es crucial para su manejo junto al uso de fármacos con actividad antimioclónica.
Introduction: Myoclonus are paroxysmal muscle contractions of short duration or abrupt loss of muscle tone, called positive and negative myoclonus respectively. A clinical case of generalized positive and negative myoclonus is presented and the aim is to describe the multiple pathophysiological mechanisms and etiologies that trigger it. Case presentation: A 35-year-old man with type 1 diabetes mellitus complicated by diabetic kidney disease on hemodialysis developed catheter-associated bacteremia due to Staphylococcus aureus and presented positive and negative myoclonus. Uremia, infection, and drugs with pro-myoclonic potential were identified as possible triggers; The incidental finding of an ischemic lesion in the caudate nucleus did not explain the semiology found in the patient. The control and removal of all the pro-myoclonic factors mentioned was carried out, along with pharmacological management with levetiracetam, thus achieving control of the symptoms. Discussion: Patients with chronic kidney disease are susceptible to the accumulation of guanidine-type toxic products, which have the potential to produce myoclonus. Furthermore, infections, the use of drugs with pro-myoclonic potential and structural lesions such as cortical ischemia are etiologies that should be considered in the differential diagnosis. The greatest impact on symptoms is observed with the control of the triggering factor and if it persists, pharmacological therapy provides good results. Conclusion: Myoclonus are relatively common movement disorders in chronic kidney disease. Identification of the trigger is crucial for its management along with the use of drugs with anti-myoclonic activity.
Subject(s)
Uremia , Cephalosporins , Renal Insufficiency, Chronic , Guanidine , Gabapentin , Levetiracetam , Analgesics, OpioidABSTRACT
Objective:To explore the effect of Toll-like receptor 9 (TLR9) signaling pathway activation on the transcriptome in the renal tubular cells.Methods:Mouse primary renal tubular epithelial cells were extracted and cultured. When the degree of cell fusion reached 80%, they were divided into two groups, which were added with 10 μL phosphate buffered saline (PBS, PBS control group) and TLR9 activator cytosine phosphate guanidine oligodeoxynucleotide (CpG-ODN) with a final concentration of 5 μmol/L (CpG-ODN treatment group). The RNA sequencing was performed on the Illumina platform after extraction. DEGseq software was used to analyze the differential expression of genes between the two groups. Goatools and KOBAS online software were used to analyze the differential genes involved signal pathways. Homer software was used to predict transcription factors.Results:Compared with the PBS control group, there were a total of 584 differentially expressed genes in the CpG-ODN treatment group, of which 102 were up-regulated and 482 were down-regulated. The most significantly enriched gene ontology (GO) terms of differentially expressed genes included response to interferon-β, defense response to virus and other inflammatory pathway. The most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways included 2'-5'-oligoadenylate synthase activity, regulation of ribonuclease activity, negative regulation of virus life cycle, cellular response to interferon-βand defense response to protozoan. The results of transcription factor prediction showed that interferon regulatory factor 3 (IRF3) was the most significantly enriched transcription factor in the promoter sequence of differential genes; the most significant transcription factor downstream of TLR9 was IRF3, and other predicted transcription factors such as transcription factor 21 (TCF21), zinc finger protein 135 (ZNF135), and PR domain containing 4 (PRDM4) might be new candidates for TLR9 signaling pathway.Conclusion:CpG-ODN activates TLR9 signaling pathway, and primary renal tubular epithelial cells can directly respond to CpG-ODN stimulation and undergo transcriptome changes, which provides a basis for further research on the molecular mechanism of TLR9 pathway in sepsis induced acute kidney injury.
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Polyhexamethyl guanidine (PHMG) is a widely used guanidine disinfectant. Since a lung injury incident induced by humidifier disinfectant (PHMG was identified as a main component) was reported in Korea, its inhalation toxicity has attracted the attention of researchers. At present, there are few domestic studies on inhalation toxicity of PHMG. In this paper, based on the domestic and foreign studies of PHMG, the toxicities of PHMG, including inhalation toxicity and associated toxic mechanism, and other potential toxicities, such as liver toxicities, cardiovascular toxicity, immunotoxicity, and reproductive and developmental toxicity, were systematically introduced, revealing that the effects of use of PHMG in a non-standard manner on the body and its possible mechanisms, which could provide reference for the use of PHMG properly and to lay a theoretical foundation and provide a scientific basis for follow-up study.
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Background: The purposes of this study were: (1) to formulate polyhexamethylene guanidine hydrochloride (PHMGH) solutions at different concentrations; (2) to evaluate their antifungal activity against a mature biofilm of Candida albicans on acrylic resins; (3) to evaluate possible effects on acrylic resins flexural strength and surface roughness. Methods: PHMGH solutions were formulated with distilled water and 0.125, 0.250, or 0.5 wt.% of PHMGH. One group without PHMGH was used as control. For antifungal activity analysis, acrylic resin specimens were contaminated with C. albicans. Specimens were immersed in PHMGH solutions or distilled water for 5 or 10 min. Ultimate flexural strength and surface roughness of acrylic resins were evaluated. Results: All PHMGH solutions at 5 or 10 min showed antifungal activity compared to the control group (p<0.05). The group with 0.5 wt.% of PHMGH showed no countable colonies regardless of the time of contact with the acrylic resins. After 10 min, all PHMGH solutions had antifungal effect without differences from 0.125 to 0.5 wt.% of PHMGH. All groups showed high flexural strength after contact with the solutions compatible with ISO 20795-1:2013 recommendation. The values of surface roughness always remained low, from 0.01 to 0.04 µm for all groups. Conclusion: Within the limitations of this study, it was concluded that the use of a solution composed by distilled water and 0.5 wt.% of PHMGH for 5 min was effective as a disinfectant agent against mature biofilm of C. albicans , maintaining acceptable roughness and flexural strength.
Antecedentes: Os objetivos deste estudo foram: (1) formular soluções de cloridrato de polihexametileno guanidina (PHM-GH) em diferentes concentrações; (2) avaliar sua atividade antifúngica contra biofilme maduro de Candida albicans em resinas acrílicas; (3) avaliar efeitos na resistência à flexão e rugosidade da superfície. Métodos: Soluções de PHMGH foram formuladas com água destilada e 0,125, 0,250 ou 0,5% em peso de PHMGH. Um grupo sem PHMGH foi usado como controle. Para análise da atividade antifúngica, amostras de resina acrílica foram contaminadas com C. albicans, e imersas em soluções de PHMGH ou água destilada por 5 ou 10 min. Resistência à flexão final e rugosidade da superfície foram avaliadas. Resultados: Todas as soluções de PHMGH aos 5 ou 10 minutos apresentaram atividade antifúngica em comparação ao grupo controle (p <0,05). O grupo com 0,5% em peso de PHMGH não mostrou colônias contáveis, independentemente do tempo. Após 10 minutos, todas as soluções de PHMGH tiveram efeito antifúngico sem diferenças de 0,125 a 0,5% em peso de PHM-GH. Os grupos apresentaram alta resistência à flexão após o contato com as soluções compatíveis com a recomendação ISO 20795-1: 2013. Os valores de rugosidade da superfície permaneceram baixos, de 0,01 a 0,04 µm para todos os grupos. Conclusão: Dentro das limitações deste estudo, concluiu-se que o uso de uma solução composta por água destilada e 0,5% em massa de PHMGH por 5 min foi eficaz como agente desinfetante contra o biofilme maduro de C. albicans, mantendo rugosidade e resistência à flexão aceitáveis.
Subject(s)
Humans , Acrylic Resins , Guanidine , Denture Bases , Antifungal Agents , Surface Properties , In Vitro Techniques , Materials Testing , Flexural StrengthABSTRACT
Subject(s)
Adult , Humans , Biopsy , Cough , Disinfectants , Dyspnea , Humidifiers , Intensive Care Units , Judgment , Korea , Lung Diseases , Lung Injury , Lung , Pathology , Pulmonary Medicine , Sputum , ThoraxABSTRACT
0.2 and p<0.05. NOTCH1 was identified as a candidate network hub gene in cases. NOTCH1 transcripts significantly increased in lung tissues from HDLI cases compared to unexposed controls (p=0.05). NOTCH1 may play an important role in pulmonary fibrosis of HDLI.
Subject(s)
Child , Humans , DNA Methylation , Gene Expression Profiling , Humidifiers , Korea , Lung Injury , Lung , Methylation , Pulmonary FibrosisABSTRACT
Disinfectants including polyhexamethylene guanidine phosphate (PHMG) and mixtures of chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) have been widely used in Korea to prevent microbial growth in the humidifier water, which triggered an outbreak of serious respiratory diseases. In addition to the respiratory syndrome, disease-related symptoms including liver toxicity, asthma, and skin allergies were also found after extensive survey of people exposed to the humidifier disinfectants (HDs). In this study, eye irritation tests were performed based on the Organization for economic co-operation and development (OECD) test guidelines 492 using EpiOcular™ which is a tissue model of reconstructed human cornea-like epithelium. As results, the raw materials of PHMG (26% as active ingredient) and CMIT/MIT (1.5% as active ingredient) were classified under UN globally harmonized system of classification and labeling of chemical (GHS) category 1 or category 2. However, aqueous dilutions of raw materials such as market products of HDs that contain 0.13% of PHMG and 0.03% of CMIT/MIT or further dilutions of the market products for humidifier that contain 0.0013% of PHMG and 0.0003% of CMIT/MIT were classified under any category, which suggested absence of eye irritation at the test concentration.
Subject(s)
Humans , Asthma , Classification , Disinfectants , Epithelium , Guanidine , Humidifiers , Hypersensitivity , Korea , Liver , Skin , United Nations , WaterABSTRACT
Disinfectants including polyhexamethylene guanidine phosphate (PHMG) and mixtures of chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) have been widely used in Korea to prevent microbial growth in the humidifier water, which triggered an outbreak of serious respiratory diseases. In addition to the respiratory syndrome, disease-related symptoms including liver toxicity, asthma, and skin allergies were also found after extensive survey of people exposed to the humidifier disinfectants (HDs). In this study, eye irritation tests were performed based on the Organization for economic co-operation and development (OECD) test guidelines 492 using EpiOcular™ which is a tissue model of reconstructed human cornea-like epithelium. As results, the raw materials of PHMG (26% as active ingredient) and CMIT/MIT (1.5% as active ingredient) were classified under UN globally harmonized system of classification and labeling of chemical (GHS) category 1 or category 2. However, aqueous dilutions of raw materials such as market products of HDs that contain 0.13% of PHMG and 0.03% of CMIT/MIT or further dilutions of the market products for humidifier that contain 0.0013% of PHMG and 0.0003% of CMIT/MIT were classified under any category, which suggested absence of eye irritation at the test concentration.
Subject(s)
Humans , Asthma , Classification , Disinfectants , Epithelium , Guanidine , Humidifiers , Hypersensitivity , Korea , Liver , Skin , United Nations , WaterABSTRACT
Several studies showed that the inflammatory and fibrotic responses induced by polyhexamethylene guanidine phosphate (PHMG-p) were similar to those observed for idiopathic pulmonary fibrosis in South Korea in 2011. “Omic” technologies can be used to understand the mechanisms underlying chemical-induced diseases. Studies to determine the toxicity of chemicals may facilitate understanding of the mechanisms underlying the development of pulmonary fibrosis at a molecular level; thus, such studies may provide information about the toxic characteristics of various substances. In this review, we have outlined the cellular and molecular mechanisms underlying idiopathic pulmonary fibrosis and described pulmonary fibrosis induced by various chemicals, including bleomycin, paraquat, and PHMG-p, based on the results of studies performed to date.
Subject(s)
Bleomycin , Epithelial Cells , Guanidine , Idiopathic Pulmonary Fibrosis , Korea , MicroRNAs , Paraquat , Pulmonary FibrosisABSTRACT
Several studies showed that the inflammatory and fibrotic responses induced by polyhexamethylene guanidine phosphate (PHMG-p) were similar to those observed for idiopathic pulmonary fibrosis in South Korea in 2011. “Omic” technologies can be used to understand the mechanisms underlying chemical-induced diseases. Studies to determine the toxicity of chemicals may facilitate understanding of the mechanisms underlying the development of pulmonary fibrosis at a molecular level; thus, such studies may provide information about the toxic characteristics of various substances. In this review, we have outlined the cellular and molecular mechanisms underlying idiopathic pulmonary fibrosis and described pulmonary fibrosis induced by various chemicals, including bleomycin, paraquat, and PHMG-p, based on the results of studies performed to date.
Subject(s)
Bleomycin , Epithelial Cells , Guanidine , Idiopathic Pulmonary Fibrosis , Korea , MicroRNAs , Paraquat , Pulmonary FibrosisABSTRACT
A total of 221 patients were evaluated to be humidifier disinfectant associated with lung injury (HDLI) through two rounds of programs through April 2015. The humidifier disinfectant (HD) brands most often associated with HDLI were found to be Oxy (n=151, 68 %) and Cefu (n=26, 17 %). Polyhexamethylene guanidine used for disinfectant for four types of HD brands including Oxy was found to be associated with the highest number of HDLI cases (n=188). Further programs are operating to identify various health effects including lung injury which may be associated with the use of HD. Not only national agencies, but also pertinent environmental health societies should cooperate in the necessary investigations so that this tragedy can be properly addressed and future incidents concerning chemicals and chemical-containing products can be prevented.
Subject(s)
Humans , Environmental Health , Guanidine , Humidifiers , Lung Injury , LungABSTRACT
An analysis of patients and fatalities due to exposure to polyhexamethylene guanidine (PHMG) shows that PHMG causes mainly lung diseases such as pulmonary fibrosis. However, no research on the other organs has been conducted on this matter yet. So, an in-depth discussion on toxicological techniques is needed to determine whether or not PHMG is toxic to organs other than just the lungs. For the test of target organ toxicity by PHMG exposure, a toxicokinetic study must first be conducted. However, measurement method for PHMG injected into the body has not yet been established because it is not easy to analyze polymer PHMG, so related base studies on analytical technique for PHMG including radio-labeling chemistry must come first. Moreover, research on exposure-biomarker and effect-biomarker must also be conducted, primarily related to clinical application. Several limitations seem to be expected to apply the biomarker study to the patient because much time has passed after exposure to the humidifier disinfectant. It is why a more comprehensive toxicological researches must be introduced to the causality for the victims.
Subject(s)
Humans , Chemistry , Guanidine , Humidifiers , Lung , Lung Diseases , Methods , Polymers , Pulmonary FibrosisABSTRACT
As part of our ongoing research on antifungal agents from Brazilian flora, eight extracts and twelve fractions from Pterogyne nitens Tul., Fabaceae, were screened for antimicrobial activity against four opportunistic fungi species (Candida albicans, Candida krusei, Candida parapsilosis and Cryptococcus neoformans) using a broth microdilution method. The present investigation reveals that P. nitens extracts and fractions were more effective against C. krusei and C. parapsilosis than against C. neoformans. The growth of C. albicans was moderately affected by all tested extracts and fractions. The strongest effects were observed for n-butanol fractions from branches (MIC = 15.6 μg/mL) and roots (MIC = 31.2 μg/mL) against C. krusei. Additionally, the chromatographic fractionation of the n-butanol fraction from branches afforded four guanidine alkaloids; N-1,N-2,N-3-triisopentenylguanidine (1), described for the first time in the Fabaceae family, and nitensidines A-C (2-4), which showed moderate activity towards C. krusei (MIC = 62.5 μg/mL) and C. parapsilosis (MIC = 31.2 μg/mL).
No contexto de nossas pesquisas por novos agentes antifúngicos obtidos da flora brasileira, oito extratos e doze frações de Pterogyne nitens Tul., Fabaceae, foram submetidos ao ensaio antifúngico pelo método de microdiluição, contra quatro espécies de fungos oportunistas, Candida albicans, Candida krusei, Candida parapsilosis e Cryptococcus neoformans. Este trabalho revelou que os extratos e frações de P. nitens foram mais ativos contra C. krusei e C. parapsilosis quando comparados a C. neoformans, sendo que o crescimento de C. albicans foi moderadamente afetado por todos os extratos e frações. As atividades mais potentes foram observadas para as frações n-butanólica dos galhos (CIM = 15,6 μg/mL) e raízes (CIM = 31,2 μg/mL) contra C. krusei. Adicionalmente, a fração n-butanólica dos galhos foi submetida ao fracionamento cromatográfico, resultando no isolamento de quatro alcaloides guanidínicos, sendo N-1,N-2,N-3-tri-isopentenilguanidina (1), descrito pela primeira vez em espécies da família Fabaceae e nitensidinas A-C (2-4), os quais apresentaram atividade antifúngica moderada contra C. krusei (CIM = 62,5 μg/mL) e C. parapsilosis (CIM = 31,2 μg/mL).
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In this study,a series of new sildenafil analogues 11-27 possessing a guanidine group were synthesized to investigate their PDE5 inhibitory activity and selectivity using[~3H]cGMP SPA kit in vitro and efficacy in the rat model of erection.Most of the compounds showed potent activity against PDE5,and more importantly,several compounds exhibited higher PDE5 selectivity over PDE6 than that of sildenafil.Structure-activity relationship of these sildenafil analogues was also discussed.Within this series of compounds,compound 15(IC_(50) =1.7 nmol/L)not only exhibited more potent PDE5 inhibitory activity than that of sildenafil (IC_(50) = 6.5 nmol/L),but also showed functional efficacy in the rat model of erection.
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DNA extraction methods for genotyping non-invasive samples have led to great advances in molecular research for ecological studies, and have been particularly useful for analyzing threatened species. However, scarce amounts of fragmented DNA and the presence of Taq polymerase inhibitors in non-invasive samples are potential problems for subsequent PCR amplifications. In this study we describe a novel technique for extracting DNA from alimentary tract cells found on external surfaces of feces and regurgitated seeds. The presence of contaminants and inhibitors is minimized and samples are preserved intact for use in other ecological research (e.g. trophic studies). The amplification efficiency and purity of the extracted DNA from feces were significantly higher than in commonly used extraction procedures. Moreover, DNA of two bird species was identified from seeds expelled by regurgitation. Therefore, this method may be suitable for future ecological studies of birds, and other vertebrate groups.
Subject(s)
Animals , Columbidae/classification , DNA, Mitochondrial/isolation & purification , Feces/chemistry , Seeds/chemistry , Columbidae/genetics , Filtration/instrumentation , Genotype , Polymerase Chain ReactionABSTRACT
AIM: To study caspase-9 expression on rat retina in the process of chronic elevation of IOP and the changes with the application of amino guanidine (AG), thus to investigate potential protective function of AG to rat retina with chronic elevation of IOP.METHODS: Immunohistochemistry, RT-PCR and Western blot were used to observe retinal morphology and expression of caspase-9 at different time points of rat with chronic IOP elevation, both affected or not affected by the application of AG.RESULTS: Compared with control group, as time passed retina of experimental group gradually had detectable morphological changes. On 21st day of chronic IOP elevation, retinas became thinner and the quantity of retinal ganglion cells (RGCs) decreased; caspase-9 expression increased, consistent with the morphological changes. The group using AG presented relatively smaller morphology changes and less expression of caspase-9.CONCLUSION: Apoptosis-related gene caspase-9 played a part in the process of chronic IOP elevation; AG protects retina by down-regulating expression of caspase-9.
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La infección fúngica invasora (IFI) está asociada a un alto índice de mortalidad, que alcanza el 50% debido a la frecuente falla en el tratamiento antifúngico. Existen dificultades para realizar un diagnóstico micológico rápido y certero dada la baja sensibilidad de los métodos convencionales, especialmente en pacientes neutropénicos y con SIDA. Numerosos métodos para diagnosticar infecciones micóticas basados en el estudio del ADN fúngico están actualmente en desarrollo. Nosotros evaluamos la utilidad de dos procedimientos de extracción y purificación del ADN fúngico presente en sangre para su posterior detección por PCR. Ambos métodos resultaron igualmente eficientes para obtener ADNs de óptima calidad y para realizar la técnica de PCR con los iniciadores universales para hongos ITS 1 e ITS 4.
Invasive fungal infections (IFI) are associated with high mortality by reaching levels of 50%, and also with a significant failure in antifungical treatments. This fact mostly obeys to difficulties in obtaining a fast and accurate mycologic diagnosis due to the low sensitivity of conventional methods, mainly in neutropenic and AIDS patients. Various methods based on fungal DNA study are currently being used for the diagnosis of mycotic infections. We herein evaluated two procedures of extraction and purification of fungal DNA in blood for their use in PCR detection. Both of them showed equal efficiency in obtaining high performance DNA with universal primers ITS 1and ITS 4 as target.
Subject(s)
Humans , DNA, Fungal/blood , DNA, Fungal/isolation & purification , Polymerase Chain Reaction , Blood Chemical Analysis/methodsABSTRACT
Objective To observe the effects of aminoguanidine and Vitamin C on lipid of streptozotocin-induced didbetic rats.Methods Diabetic nephropathy rats were induced by intraperitoneal injection of streptozotocin.Fifty Sprague-Dawley(SD) rats were randomly divided into normal control group,model group,Vitamin C group,aminoguanidine group and Vitamin C associated with aminoguanidine group.All the rats were treated for 16 weeks.During and after the treatment,the general state of the rats was observed and the levels of blood sugar,triglyceride,total cholesterol,low density lipoprotein,high density lipoprotein,glycosylated hemoglobin,and glycated low density lipoprotein in different groups were detected.Results The rats in the four groups suffered diabetes mellitus and renal function lesion.Aminoguanidine and Vitamin C could improve the general state of the rats,but had no effect on the blood sugar.Vitamin C could decrease the levels of triglyceride,serum cholesterol,low density lipoprotein,glycosylated hemoglobin,and glycated low density lipoprotein,and increase the level of high density lipoprotein.There was a synergistic effect with the combination of the two drugs. Conclusion Aminoguanidine and Vitamin C have no effect in the decrease of blood sugar level but have a certain effect on regulating blood lipid.There is a synergistic effect with the combination of the two drugs.
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The effect of urea, guanidine hydrochloride and sodium dodecyl sulphate on glycinin, the high molecular weight protein fraction from soybean has been investigated by analytical ultracentrifugation. Urea and guanidine hydrochloride dissociate the protein to a '2S' protein through the intermediary 7S and 4S proteins. However, in sodium dodecyl sulphate the protein directly dissociates to a 2S protein. Analysis of the data by calculation of per cent fraction and S20,w value indicates that dissociation and denaturation of glycinin occur simultaneously in the presence of the above reagents but to different extents.
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The interaction of α-globulin with urea/guanidine hydrochloride was investigated by determining the apparent partial specific volumes of the protein in these solvents. The apparent partial specific volumes were determined both under isomolal and isopotential conditions. The preferential interaction parameter with solvent components calculated were 0.08 and 0.1 g of urea and guanidine hydrochloride respectively per g protein. In both the cases the interaction was not preferential with water. The total binding of denaturant to α-globulin was calculated both for urea and guanidine hydrochloride and the correlation between experimentally determined number of mol of denaturant bound per mol of protein and the total number of peptide bonds and aromatic amino acids were found to be in excellent agreement with each other. The changes in volume upon transferring α-globulin from a salt solution to 8 Μ urea and 6 Μ guanidine hydrochloride were also calculated.