BACKGROUND: Our goal was to evaluate anti-calcification effects of decellularization and diverse fixing methods including preincubation of the bovine pericardium with ethanol. We also assessed changes in mechanical properties. MATERIAL AND METHOD: Harvested bovine pericardium was decellularized with 0.25% sodim dodecysulfate and then treated with 5 methods of fixation: (1) 0.5% glutaraldehyde (GA) for 14 days, (2) 0.5% GA for 5 days, 2% GA for 2 days and 0.25% GA for 7 days, (3) 0.5% GA for 5 days, 2% GA for 2 days, 0.25% GA for 7 days, and then 70% ethanol for 2 days, (4) 0.5% GA for 5 days, a mixture of 2% GA and 70% ethanol for 2 days, and 0.25% GA for 7 days, (5) 0.5% GA for 5 days, a mixture of 2% GA, 65% ethanol, and 5% octanediol for 2 days and then 0.25% GA for 7 days. All treated bovine pericardia were tested for histological variables, lipid content, and mechanical properties including tensile strength and thermal stability. A total 10 kinds of differently treated bovine pericardia were implanted into rat subdermis and harvested 8 weeks later. Harvested pericardia were evaluated for calcium content. RESULT: No protein denaturation was observed microscopically after decellularization. There was a 32% mean decrease in tensile strength index after decellularization in the bovine pericardium group fixed. Octanediol preincubation attenuated the decrease in tensile strength and maintained thermal stability. TG and cholesterol were not affected by decellularization but were decreased by organic solvent. Calcium content was decreased after decellularization, and organic solvent preincubation decreased calcification in the non-decellularized bovine pericardium group. CONCLUSION: Decellularization and organic solvent preincubation have anti-calcification effects but decellularization may cause mechanical instability. A method of decellularization and fixation that does not cause damage to matrices will be needed for evaluation of the next step in using tissue-engineering for replacement of cardiac valves.
Animals ; Calcium ; Cholesterol ; Ethanol ; Glutaral ; Heart Valves ; Pericardium ; Protein Denaturation ; Rats ; Tensile Strength ; Tissue Engineering

OBJECTIVETo characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation.

METHODSThe refolding process of recombinant bovine β-lactoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro.

RESULTSRenaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.

CONCLUSIONThe degree of protein renaturation correlated with the IgE-binding capacity of the protein. Results from this study may be of help for food allergy therapy and development of vaccination in the future.

Allergens ; Animals ; Cattle ; Circular Dichroism ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulin E ; Lactoglobulins ; chemistry ; metabolism ; Models, Molecular ; Protein Binding ; Protein Conformation ; Protein Denaturation ; Protein Folding ; Spectrometry, Fluorescence ; methods

BACKGROUND: Inducible heat shock protein 70s (iHSP70) are expressed by stressful stimuli that result in protein denaturation, and are thought to assist in the maintenance of cellular integrity and viability. In addition, iHSP70 is known to be a sensitive marker of neuronal injury. To my best knowledge, no previous studies have been documented on iHSP70 induction by nociceptive impulse transmission through peripheral nerves not by direct neural damage. The purpose of this study was to examine the hypothesis that iHSP70 can be expressed in the nervous system, which is related to the dental nociceptive pathway, by tooth pulp inflammation. METHODS: The pulp of rat mandibular molars was exposed. Animals were sacrificed at 1, 4, and 7 days after pulpal exposure, and the pulps were evaluated histologically. Also, iHSP70 levels were examined in the Gasserian ganglion (GG) and the trigeminal sensory nucleus (TSN). RESULTS: At 4 days after pulpal exposure, iHSP70 was significantly more expressed in the ipsilateral GG than in the contralateral GG. In the histological study, inflammation was found in the entire pulp tissue at 4 days. There were no significant differences in iHSP70 levels between the ipsilateral TSN and the contralateral TSN. Also, there were no significant differences in iHSP70 expression of GG and TSN between both sides at 1 and 7 days after pulpal exposure. CONCLUSIONS: These results suggest that iHSP70 can be expressed in the GG at 4 days after pulpal exposure by nociceptive impulses due to pulpal inflammation.
Animals ; Heat-Shock Proteins* ; Hot Temperature* ; HSP70 Heat-Shock Proteins* ; Inflammation ; Molar ; Nervous System ; Neurons ; Peripheral Nerves ; Protein Denaturation ; Rats* ; Tooth ; Trigeminal Ganglion

BACKGROUND: Phenol has effects like surgical neurectomy, but may evoke pain after local infiltration in nerves. Transection of peripheral nerves may induce neuropathic pain through increased spontaneous discharge and other mechanisms. Proto-oncogene, c-fos, is an indicator of neuronal activity, and its expression in the spinal cord may be related to pain development, because inhibition of c-fos expression has corresponding effects like analgesia. We evaluated the effects of local infiltration of phenol and transection injury at the sciatic nerve on c-fos expression in the spinal cords of rats. METHODS: Sixteen male Sprague-Dawley rats were divided into 2 groups; transection of the sciatic nerve was performed for group 1; phenol was infiltrated into the sciatic nerve in group 2. Three hours, 1 week, 2 weeks, and 3 weeks after the experiment, the corresponding spinal cord was stained immunohistochemically for c-fos. RESULTS: c-fos was expressed from 3 hours to 2 weeks over the laminae of the dorsal horn in each group. Phenol increased the expression of c-fos initially, but decreased 1 week later. Transection injury did not increase it initially, but showed the peak expression at 1 week, and maintained it for 2 weeks. Therefore, it seems that phenol, rather than the transection injury, stimulates c-fos expression early, but decreases later. CONCLUSIONS: Phenol treatment, caused by chemical block due to protein denaturation and nonspecific inflammation, may induce less neuropathic pain than the transection of a nerve.
Analgesia ; Animals ; Horns ; Humans ; Inflammation ; Male ; Neuralgia ; Neurons ; Peripheral Nerves ; Phenol* ; Protein Denaturation ; Proto-Oncogenes ; Rats* ; Rats, Sprague-Dawley ; Sciatic Nerve* ; Spinal Cord*

As a virus-like particle, hepatitis B surface antigen (HBsAg) was the primary component of hepatitis B vaccine. HBsAg was maintained by the non-covalent interaction of proteins and lipids. The intact structure of HBsAg particle was vital to its function. However, there was no report about the effects of solvent environment on HBsAg structure. In this paper, we studied the effects of temperature, pH, ionic type and salt concentration on HBsAg structure. The results showed that HBsAg was stable at normal temperature, but began to denature above 60 degrees C. The aggregation of HBsAg at pH 3.0 and 4.0 was nearly irreversible, but partly reversible at pH 5.0. The influence of ionic type on HBsAg was generally in accordance with Hofmeister sequence, except that SO4(2-) caused more aggregation than F-. HBsAg aggregates started to be visible in 0.4 mol/L (NH4)2SO4, and the extent of aggregation increased with the salt concentration. Therefore, caution must be taken when using (NH4)2SO4 in the hydrophobic chromatography purification of HBsAg.
Ammonium Sulfate ; chemistry ; Hepatitis B Surface Antigens ; chemistry ; Hepatitis B Vaccines ; chemistry ; Hepatitis B virus ; chemistry ; Hydrogen-Ion Concentration ; Protein Denaturation ; Solvents ; Temperature

To investigate the effects of induction temperature on the expression product and the impact of urea concentration on the purification, HIV-1 Gag inclusion bodies from E. coli induced at 30 degrees C (IB30) and 37 degrees C (IB37) were dissolved with urea of different concentrations. The solubility and yield of refolding were compared. IB30 were dissolved with 2 mol/L and 8 mol/L urea, and then purified with chromatography. IB30 were found easier to be solubilized in low concentration of urea and easier to be refolded than IB37. Furthermore, compared to the IB30 dissolved in 8 mol/L urea, Gag protein solubilized in 2 mol/L urea was purified to higher purity with gel filtration (GF) and ion exchange (IEX) chromatography. Gag inclusion body induced at lower temperature may contain more protein with native-like or reversibly-denatured structures, and solubilization in the presence of low concentrations of urea can help to retain these structures. This study has provided new insights into the purification of proteins from inclusion bodies.
Escherichia coli ; genetics ; metabolism ; HIV-1 ; genetics ; Humans ; Inclusion Bodies ; metabolism ; Protein Denaturation ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Temperature ; Urea ; chemistry ; gag Gene Products, Human Immunodeficiency Virus ; biosynthesis ; genetics

OBJECTIVETo study the clinical value of muscle autograft denatured by microwave for repair of gap in removal of facial neuroma.

METHODSA case of patient with a larger facial nerve Schwann cell neuroma was reported. Based on the good results of our experimental research on rats recently, we completed the operation by transmastoid approach for removal of facial neuroma, and the gap of the nerve was repaired with muscle autograft denatured by microwave.

RESULTSThe patient was followed up for one year, and her facial function recovery on the affected side was satisfactory.

CONCLUSIONMuscle autograft denatured by microwave technique is convenient, highly efficient for repairing facial nerve gap after removal of facial neuroma.

Adult ; Facial Neoplasms ; complications ; surgery ; Facial Nerve ; physiology ; Facial Paralysis ; etiology ; surgery ; Female ; Follow-Up Studies ; Humans ; Microwaves ; Muscle, Skeletal ; transplantation ; Nerve Regeneration ; Neurilemmoma ; complications ; surgery ; Protein Denaturation ; Transplantation, Autologous

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.
Citrate (si)-Synthase ; chemistry ; Cloning, Molecular ; Conserved Sequence ; Escherichia coli ; cytology ; enzymology ; GTP Phosphohydrolases ; chemistry ; genetics ; isolation & purification ; metabolism ; Guanosine Diphosphate ; pharmacology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Molecular Chaperones ; chemistry ; genetics ; isolation & purification ; metabolism ; Protein Denaturation ; drug effects ; Protein Renaturation ; drug effects ; Ribosomes ; metabolism ; alpha-Glucosidases ; chemistry

Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.
Animal ; B-Lymphocytes/physiology* ; B-Lymphocytes/drug effects ; Bone Marrow/metabolism ; Cell Division/physiology ; Chromatography, High Pressure Liquid ; Chymotrypsin/pharmacology ; Dose-Response Relationship, Drug ; Growth Substances/pharmacology* ; Growth Substances/chemistry ; Heat ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Molecular Weight ; Polymyxin B/pharmacology ; Protein Denaturation ; Spleen/metabolism* ; Thymus Gland/metabolism ; Trypsin/pharmacology