Chaperone-like effect of the linker on the isolated C-terminal domain of rabbit muscle creatine kinase.

Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.

[Tumors of different histological type in unilateral salivary glands: a case report].

Tumors of synchronous benign and malignant in unilateral salivary glands have rarely been reported. A case of 21-year-old girl who was diagnosed as synchronously adenoid cystic carcinoma of the left parotid and pleomorphic adenoma of the left submandibular gland. The classification, clinic pathology, diagnosis, possible mechanism were discussed based on similar literatures.

[Function of P63 on the development of salivary glands].

OBJECTIVE: To study the expression of P63 in human salivary gland development and the existing of salivary gland stem cells. METHODS: 24 embryonic salivary gland samples in different stage, 10 normal salivary gland samples were collected. HE-stained and immunochemistry stain were used. RESULTS: It could be seen on the HE-stained sections that the epithelial buds proliferated to form the epithelial branches and duct systems, finally the terminal cells differentiated into ductal, myoepithelial and acinous. During the development of salivary gland, the expression of P63 was gradually reduced. In normal adult salivary gland samples, the positive cell interspersed in the basal layer of intercalated duct, secretory duct and excretory duct. CONCLUSION: P63 plays an important role in human salivary gland development. The result of our experiment shows the distributive characteristic of salivary gland stem cells, which exist abroad in bud stage, but decrease and only exist in the basal layer of ducts in normal adult salivary gland.

[Effect of focal adhesion kinase on biological characteristics of Tca8113 tongue squamous cell carcinoma].

OBJECTIVE: To investigate the effect of FAK on biological characteristics of tongue squamous cell carcinoma cell Tca8113. METHODS: Lipofect was used to transfect FAK into Tca8113 cell lines. The difference in expression of FAK between the transfected and non-transfected cell lines was detected by immunofluorescence assay. Then by use of transwell room and wash way, the difference in invasion and adhesion ability between the transfected and non-transfected cell lines was tested. MTT method was adopted in finding the change of proliferation. RESULTS: RT-PCR analysis showed that the FAK gene was downregulated by Tca8113 cells. FAK expression was significantly down regulated by the transfection of FAK ASODN (P<0.05). Antisense FAK ODN significantly inhibited proliferation, adhesion and the invasion of Tca8113 cells. CONCLUSION: FAK plays an important role on biological characteristics of tongue squamous cell carcinoma cell Tca8113.

Role of the linker between the N- and C-terminal domains in the stability and folding of rabbit muscle creatine kinase.

Domain-domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of the linker in the folding, stability and function of multidomain proteins. In this research, muscle creatine kinase (CK), a dimeric two-domain protein, was used as a model protein to investigate the role of the linker in CK activity, stability and folding by mutational analysis. Two of the three mutations, L115D and L121D, resulted in a gradual decrease in CK activity and secondary structures, but did not affect CK inactivation induced by heat or guanidine hydrochloride (GdnHCl). The mutations also caused much more serious aggregation during heat- and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, none of the three mutants could successfully recover their activities by dilution-initiated refolding, and the rate constant of CK refolding was gradually decreased by the mutations. These results suggested that mutations of the hydrophobic residues in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the two domains. The results herein indicated that in addition to its role in the in vivo functions, the linker also played a crucial role in the stability and folding of CK.

Exploration of the polar microenvironment around the reactive cysteine in rabbit muscle creatine kinase.

The polar microenvironment around the reactive Cys283 of rabbit muscle creatine kinase was explored using kinetic analysis of substrates reaction in the presence of modifiers. In the present study, three specific sulphydryl reagents, 5,5'-dithiobis(2-nitrobenzoic acid), 6,6'-dithiodinicotinic acid and 2,2'-dithiodipyridine, were applied as modifiers to react with Cys283 of creatine kinase. The inactivation kinetics of creatine kinase by the modifiers was analyzed. The microscopic rate constants for reactions of the modifiers with free enzyme and enzyme-substrate complexes were also determined. The results suggested that the inactivation rate of creatine kinase by 5,5'-dithiobis(2-nitrobenzoic acid) was the fastest, followed by 6,6'-dithiodinicotinic acid and then 2,2'-dithiodipyridine. Interestingly, 5,5'-dithiobis(2-nitrobenzoic acid) and 6,6'-dithiodinicotinic acid functioned as non-complexing modifiers, while 2,2'-dithiodipyridine did a complexing modifier. The results here indicated that the electrophilic group was predominant around Cys283, and that the presence of substrates seemed to have different effects on the inactivation reactions of creatine kinase by the three modifiers. Furthermore, the findings in this study may provide a novel explanation for the low pKa value of Cys283.

Sodium barbital is a slow reversible inactivator of rabbit-muscle creatine kinase.

As a depressant of the central nervous system, the clinical effect of sodium barbital has been extensively studied. Here we report on sodium barbital as an inhibitor of rabbit-muscle creatine kinase (CK), which plays a significant role in energy homeostasis in the muscles. Although sodium barbital gradually inhibits the activity of CK with increased concentration, the inhibition effect can be completely reversed by dilution, indicating that the inactivation process is reversible. Detailed kinetics analysis, according to a previously presented theory, indicates that sodium barbital functions as a non complexing inhibitor, and its inhibition effect on CK is a slow reversible inactivation. In this study, a kinetic model of the substrate reaction is presented, and the microscopic rate constants for the reaction of sodium barbital with the free enzyme and the enzyme-substrate complexes are determined. Kinetic analysis reveals that sodium barbital might compete with both creatine and ATP, but mainly with creatine, to inhibit the activity of CK. The results suggest that CK might be a target for sodium barbital in vivo.

[Expression of deltaNp63 in salivary gland tumours].

OBJECTIVE: To detect the expression of deltaNp63 in human salivary gland tumor and analyze its role in the malignant salivary gland tumors. METHODS: Collecting the samples from 68 cases of pathologically proven salivary gland tumours and investigating the microscopic sections with HE stain and immunohistochemical-SP stain. RESULTS: It was found that the expression of deltaNp63 is gradually increased in the malignant tumours, the myoepithelial cells and the basal cells of the salivary gland tumors are positive, and the expression of deltaNp63 is correlated directly with benign and malignant tumor. CONCLUSION: deltaNp63 plays an important role in salivary gland tumours. deltaNp63 possesses some special activity that is characteristic of oncogene. p63 is a sensitive and highly specific marker of myoepithelial cells in salivary gland tumours and an additional marker for defining myoepithelial histogenesis. p63 is of definite clinical value in falicitating diagnosis and differential diagnosis.

Sequential events in ribonuclease A thermal unfolding characterized by two-dimensional infrared correlation spectroscopy.

The conformational changes in the thermal denaturation of bovine pancreatic ribonuclease A was followed with infrared spectra and analyzed by second derivative and two-dimensional correlation techniques. By analyzing the sequential events in each transition stage, the results were consistent with a step-wise thermal denaturation mechanism in which the structural adjustment of the N-terminal and the opening of the central structure of the protein come before the main unfolding process. Non-native turns were found to form along with the unfolding of the native structures. The central region that is composed of some beta-sheet and alpha-helical structures was found to be the most stable part that might form the residual structure at high temperatures.

Oligomerization and aggregation of bovine pancreatic ribonuclease A: characteristic events observed by FTIR spectroscopy.

Nonnative protein aggregation, which is a common feature in biotechnology, is also a clinical feature in more than 20 serious degenerative diseases. We studied the specific events of bovine pancreatic ribonuclease A thermal aggregation by a combination of second derivative infrared analysis and two-dimensional infrared correlation spectroscopy. By comparing the events that occur in reversible and irreversible thermal unfolding processes, certain events that were related to protein aggregation were characterized. Particularly, a band that appeared at high temperatures was assigned to the cross beta-structures in oligomers. The effect of pH, NaCl, and ethanol on ribonuclease A oligomerization as well as further aggregation induced by heat were studied and dissimilar effects of these additives were found. Basic pH and NaCl could accelerate the thermal aggregation but did not affect the formation of oligomers, whereas ethanol could increase both the aggregation rate and the population of oligomers. Our results suggested that the aggregation of RNase A might be initiated by hydrophobic interactions, controlled by oligomerization and mediated by electrostatic interactions. Moreover, the strategy of using second derivative and two-dimensional infrared analysis might provide a potential powerful tool to study the events that are directly related to the initiation of protein aggregation.

[Expression and clinical significance of focal adhesion kinase in oral squamous cell carcinoma].

OBJECTIVE: To study the correlation between expression levels of focal adhesion kinase (FAK) in oral squamous cell carcinoma (OSCC) and clinical prognosis with pathologic parameters. METHODS: Eighty paraffin embedded tissue specimens of OSCC were studied by immunohistochemistry. The data was analyzed by SPSS statistical software. RESULTS: FAK was highly expressed in OSCC tissues, in contrast to none or a low expression in normal oral epithelial tissue, and the heterogeneous staining was mainly located in the cell membrane and cytoplasm. FAK overexpression significantly correlated with histological differentiation and TNM stage, lymphatic metastasis. Multivariate analysis revealed that overexpression of FAK was the independent prognostic factor. CONCLUSION: The overexpression of FAK in the peripheral area of OSCC may serve as a useful biological factor affecting prognosis.

[Clinicopathological study of Ewing's sarcoma/primitive neuroectodermal tumor in oral and maxillofacial region].

OBJECTIVE: To study the clinicopathological features and diagnostic criteria of Ewing's sarcoma/primitive neuroectodermal tumor (Ewing's sarcoma/PNET) in oral and maxillofacial region. METHODS: There were 15 patients with Ewing's sarcoma/PNET in the last 35 years at our hospital, aged 1-49 years and mean 14.5 years. The most common manifestation was swelling of the affected region. The cases were analyzed and histological and immunohistochemical studies were also conducted to examine CD99 (12E7), Vimentin, NSE, S-100, Syn, CD45 (LCA), desmin. RESULTS: (1) The most common histological pattern of Ewing's sarcoma/PNET was a lobular arrangement of uniform, small, hyperchromatic cells in a fibrous background. Some of these tumors were rich in cytoplasmic glycogen. (2) Of the 7 cases, IHC was positive for CD99 (12E7) and Vimentin and negative for lymphoid (CD45), muscle (desmin) markers. S-100 was positive in 4 cases and NSE negative in 3, Syn positive in 1. NSE, S-100, Syn were all negative in 2 cases. CONCLUSIONS: Ewing's sarcoma/PNET is more common in teenagers and young people. Immunohistochemistry is essential to distinguish Ewing's sarcoma/PNET from other small round cell tumors. Immunohistochemistry is useful in the diagnosis.

Conformational change in the C-terminal domain is responsible for the initiation of creatine kinase thermal aggregation.

Protein conformational changes may be associated with particular properties such as its function, transportation, assembly, tendency to aggregate, and potential cytotoxicity. In this research, the conformational change that is responsible for the fast destabilization and aggregation of rabbit muscle creatine kinase (EC 2.7.3.2) induced by heat was studied by intrinsic fluorescence and infrared spectroscopy. A pretransitional change of the tryptophan microenvironments was found from the intrinsic fluorescence spectra. A further analysis of the infrared spectra using quantitative second-derivative and two-dimensional correlation analysis indicated that the changes of the beta-sheet structures in the C-terminal domain and the loops occurred before the formation of intermolecular cross-beta-sheet structures and the unfolding of alpha-helices. These results suggested that the pretransitional conformational changes in the active site and the C-terminal domain might result in the modification of the domain-domain interactions and the formation of an inactive dimeric form that was prone to aggregate. Our results highlighted the fact that some minor conformational changes, which were usually negligible or undetectable by normal methods, might play a crucial role in protein stability and aggregation. Our results also suggested that the changes in domain-domain interactions, but not the dissociation of the dimer, might play a crucial role in the thermal denaturation and aggregation of this dimeric two-domain protein.

Osmophobic effect of glycerol on irreversible thermal denaturation of rabbit creatine kinase.

Protein stability plays an extremely important role not only in its biological function but also in medical science and protein engineering. Osmolytes provide a general method to protect proteins from the unfolding and aggregation induced by extreme environmental stress. In this study, the effect of glycerol on protection of the model enzyme creatine kinase (CK) against heat stress was investigated by a combination of spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal inactivation and aggregation in a concentration-dependent manner. The spectroscopic measurements suggested that the protective effect of glycerol was a result of enhancing the structural stability of native CK. A further thermodynamic analysis using the activated-complex theory suggested that the effect of glycerol on preventing CK against aggregation was consistent with those previously established mechanisms in reversible systems. The osmophobic effect of glycerol, which preferentially raised the free energy of the activated complex, shifted the equilibrium between the native state and the activated complex in favor of the native state. A comparison of the inactivation rate and the denaturation rate suggested that the protection of enzyme activity by glycerol should be attributed to the enhancement of the structural stability of the whole protein rather than the flexible active site.

[A retrospective study on 615 cases of minor salivary gland tumors].

OBJECTIVE: To analyse the clinical and pathological characteristics of minor salivary gland tumors. METHODS: A retrospective analysis on 615 cases of intraoral minor salivary gland tumors from 1990-2002 with a confirmed pathologic diagnosis was carried out. RESULTS: In 615 cases of minor salivary gland tumors, 265 cases were benign, 350 cases malignant. Pleomorphic adenoma was the most common entity and accounted for 81.1% of all benign tumors. Adenoid cystic carcinoma comprised 32.9% of the malignant sample and was the most frequent malignant tumor. The principal location was the palate. Female was a little more of the benign and a male prevalence was observed on the malignant tumor. The mean age of patients with benign and malignant tumors were 40.9 and 49.1 years old, respectively (P < 0.05). CONCLUSION: We consider extraordinary manifestation as histopathology, tumor, primary location, age and sex, presenting the results of a review of our experience with those minor salivary gland tumors.

Protein thermal aggregation involves distinct regions: sequential events in the heat-induced unfolding and aggregation of hemoglobin.

Protein thermal aggregation plays a crucial role in protein science and engineering. Despite its biological importance, little is known about the mechanism and pathway(s) involved in the formation of aggregates. In this report, the sequential events occurring during thermal unfolding and aggregation process of hemoglobin were studied by two-dimensional infrared correlation spectroscopy. Analysis of the infrared spectra recorded at different temperatures suggested that hemoglobin denatured by a two-stage thermal transition. At the initial structural perturbation stage (30-44 degrees C), the fast red shift of the band from alpha-helix indicated that the native helical structures became more and more solvent-exposed as temperature increased. At the thermal unfolding stage (44-54 degrees C), the unfolding of solvent-exposed helical structures dominated the transition and was supposed to be responsible to the start of aggregation. At the thermal aggregation stage (54-70 degrees C), the transition was dominated by the formation of aggregates and the further unfolding of the buried structures. A close inspection of the sequential events occurring at different stages suggested that protein thermal aggregation involves distinct regions.

Two-dimensional infrared correlation spectroscopy study of sequential events in the heat-induced unfolding and aggregation process of myoglobin.

Unfolding and aggregation are basic problems in protein science with serious biotechnological and medical implications. Probing the sequential events occurring during the unfolding and aggregation process and the relationship between unfolding and aggregation is of particular interest. In this study, two-dimensional infrared (2D IR) correlation spectroscopy was used to study the sequential events and starting temperature dependence of Myoglobin (Mb) thermal transitions. Though a two-state model could be obtained from traditional 1D IR spectra, subtle noncooperative conformational changes were observed at low temperatures. Formation of aggregation was observed at a temperature (50-58 degrees C) that protein was dominated by native structures and accompanied with unfolding of native helical structures when a traditional thermal denaturation condition was used. The time course NMR study of Mb incubated at 55 degrees C for 45 h confirmed that an irreversible aggregation process existed. Aggregation was also observed before fully unfolding of the Mb native structure when a relative high starting temperature was used. These findings demonstrated that 2D IR correlation spectroscopy is a powerful tool to study protein aggregation and the protein aggregation process observed depends on the different environmental conditions used.