Evaluation of lactate dehydrogenase enzyme activity in saliva and serum of oral submucous fibrosis patients.

BACKGROUND: Of all oral precancerous conditions, Oral Submucous Fibrosis is of greater concern because of its disabling nature and relative greater chances of malignant transformation. This malignant transformation involves glycolytic pathways that can alter lactate dehydrogenase levels. Therefore the aim of this study was to estimate the LDH levels in saliva and serum of subjects with OSMF and to compare them with healthy controls and to correlate the relationship between pathogenesis of OSMF and the LDH enzyme. METHODS: Sixty Subjects were recruited for this study and divided into two groups, 30 subjects with OSMF (Group A) and 30 healthy controls (Group B). Venous blood and unstimulated whole saliva measuring 1 ml was collected from each of these evaluated for LDH levels using the standard kit method. The data obtained were subjected to statistical analysis using the SPSS software version 17. RESULTS: The average salivary LDH value for Group A was 606.83 ± 60.09 U/l and for Group B was 80.73 ± 20.06 U/l. salivary LDH was greater in group A than Group B and this was statistically significant. On comparing the serum and salivary LDH in Group A with the clinical staging of OSMF, the results were not statistically significant. Similarly no statistically significant relationship was found on comparing the serum and salivary LDH in Group A (OSMF subjects) with duration of habit. CONCLUSION: This study provides additional rationale for the role of salivary LDH in the early diagnosis and prognosis of oral submucous fibrosis.

Surgical correction of excessive gingival display in class I vertical maxillary excess: Mucosal strip technique.

There are several conditions that results in excessive gingival display. In case of class I vertical maxillary excess the reason for this excessive display is the hypermobile lip. Though orthodontic treatment is the choice of treatment, surgical repositioning along with the orthodontics gives more predictable and stable results. This case report discusses cosmetic surgical management of case with class I vertical maxillary excess with excessive gingival display. The technique involves removal of strip of mucosal tissue from the labial vestibule thereby limiting the retraction of elevator muscles.

Giant cell fibroma: A clinicopathological study.

OBJECTIVE: Giant cell fibromas (GCF) of the oral cavity are found predominantly in Caucasians and rarely in other races. This retrospective study was done to evaluate the clinicopathological features of GCFs in a sample of Indian population. MATERIALS AND METHODS: 21 oral GCF cases were investigated from the year 1995 to 2010. Clinical data and microscopic features were reviewed and analyzed. RESULTS: The mean age of patients at the time of diagnosis was 39years. Oral GCF occurred in patients between 6 and 67 years of age. The lesions were 4-17 mm in greatest dimension. GCF frequently has the provisional diagnosis of fibroma or papilloma. All tumors were treated by total surgical excision and no recurrence was reported. The consistent and diagnostic feature was the presence of large stellate giant cells, usually with one or two nuclei. Multinucleated giant cells were seen occasionally. These giant cells were most numerous in the connective tissue beneath the epithelium. CONCLUSION: Though there are distinct histopathologic features for GCF, its clinical presentation and prognosis are similar to the conventional fibroma/fibroepithelial polyp.

Cognitive assessment and health education in children from two different cultures.

This paper presents research aimed at investigating high level comprehension and problem solving processes in children in two different countries, India and Colombia. To this end, we use a series of health-related cognitive tasks as assessment tools. In one study, we also examine children's performance on these cognitive tasks, in relation to their nutritional status and parasitic load. The ages of the children tested ranged from 2 through 14 years. The tasks were designed to assess comprehension of sequences, organization of concepts, understanding of health routines (hygiene practices) and evaluation of hypothesis and evidence. The results show that children approach the different tasks with a baggage of beliefs and local knowledge of the world which determines their reasoning process, their comprehension and their problems solving. The results are discussed in terms of cognitive assessment approaches, as applied to classroom instruction. Given that children construct their understanding of reality based on what they already know and that education does not take this into account, we recommend that assessment tools should be devised that can tap prior knowledge and understanding, such that this can be analyzed and understood in relation to knowledge taught in the classroom. Current educational assessment fails in such an endeavor.

Inhibition of tumor cell mitochondrial respiration by macrophage cytotoxic mediators distinct from interferon-gamma.

Macrophage-mediated inhibition of mitochondrial respiration in EMT-6 murine mammary adenocarcinoma cells can be mimicked in vitro by treatment of the cells with interferon-gamma (IFN-gamma) in combination with tumor necrosis factor, interleukin-1, or lipopolysaccharide. Conditioned supernatants obtained from activated macrophages appear to contain interferon-gamma, suggesting that inhibition of mitochondrial respiration in tumor cells was caused by synergy of IFN-gamma with other cytokines. To further characterize monokines that cause inhibition of mitochondrial respiration in tumor cells, EA13.5 macrophage-like cells were isolated and selected for inhibition of mitochondrial respiration in EMT-6 tumor cells. After stimulation with IFN-gamma and lipopolysaccharide, the EA13.5 cells released into conditioned supernatants a cytotoxic mediator that induced nitric oxide synthesis and caused lesions in the electron transport chain of EMT-6 cells similar to the lesions caused by activated peritoneal macrophages. Enzyme-linked immunosorbent assay demonstrated that the conditioned supernatants produced by EA13.5 macrophage cells did not contain IFN-gamma. Treatment of the EA13.5 cell-conditioned supernatants with neutralizing antibody against IFN-gamma did not abrogate the inhibition of mitochondrial respiration in EMT-6 cells caused by these conditioned supernatants. This study demonstrated that unidentified macrophage cytotoxic mediators distinct from IFN-gamma are involved in the induction of nitric oxide synthesis and inhibition of mitochondrial respiration in tumor cells.

Independence of the pattern of early cytokine release from autoregulation by nitric oxide.

The L-arginine-dependent tumor cell cytotoxicity produced by activated macrophages (M phi) may be mediated either directly by production of nitric oxide (NO), or by induction of NO synthesis in the tumor cell. The influence of M phi NO synthesis on the release of soluble cytotoxic mediators was investigated in this study. The synthesis of M phi NO, measured as nitrite, was detected 6 h after lipopolysaccharide (LPS)-triggering and reached a peak level by 44 h. A concurrent decrease in M phi viability beginning at 18 h after triggering was detected during a period of 72 h in culture. Both the production of NO and loss of viability correlated with the presence of L-arginine in the incubation media and was inhibited by NG-monomethyl-L-arginine (NMA). The medium in which LPS-triggered adherent peritoneal exudate cells were incubated was examined for the presence of tumor necrosis factor (TNF), gamma interferon (IFN-gamma), and the soluble mediators that induce mitochondrial respiratory inhibition in tumor cells. All of these effector molecules were released at peak levels into the conditioned supernatants within 12 h after LPS-triggering. The peak level obtained for each effector molecule was influenced by the media in which the M phi was incubated; however, no correlation was detected between the level of cytokines produced and the synthesis of nitrite by the M phi indicating that NO synthesis has no inhibiting effect on the initial burst of cytotoxic factors released.

Proximity and ligand-induced movement of interdomain residues in myosin subfragment 1 containing trapped MgADP and MgPPi probed by multifunctional cross-linking.

The conformations of myosin subfragment 1 containing trapped MgADP or MgPPi have been studied by investigating the spatial disposition of the remainder of the subfragment 1 structure to the covalently bridged ATPase-related thiols SH1 and SH2. This has been done by synthesizing a trifunctional photoactivatable reagent 4,4'-bis(N-maleimido)benzophenone and reacting it with subfragment 1 in the presence of these ligands. Modification of subfragment 1 by this reagent mimics closely the changes in the ATPase properties as noted previously for modification with p-phenylenedimaleimide. In addition, noncovalent trapping of nucleotide also results, presumably by the bridging of the SH1 and SH2 thiols. On photolysis, cross-linking from the reagent bridging the thiols to other regions in subfragment 1 can be observed, but the extent and course of the photoinduced cross-linking depend on the nature of the trapped ligand. For subfragment 1 with trapped MgADP, a high efficiency cross-linking occurs between the 21-kDa segment and the 50-kDa segment. With MgPPi as the trapped ligand, low efficiency cross-linking occurs between the bridged thiols and either the 27-kDa N-terminal or the 50-kDa segments of the heavy chain. These results indicate that without the adenosine moiety, the binding of MgPPi to subfragment 1 leaves the protein in a flexible state so that residues in both the 27-kDa and the 50-kDa segment can move within the cross-linking span of the activated benzophenone triplet. The trapping of MgADP apparently results in a more rigid state for the subfragment 1 in which residues in the 50-kDa segment are spatially close to the bridged thiols, thus enabling photocross-linking to proceed with higher efficiency.

Substructure of skeletal myosin subfragment 1. Preferential destabilization of a domain by methanol and its effect on catalytic activity.

Evidence is presented that in increasing concentrations of methanol the structure of the subfragment 1 is perturbed in such a way that the Mr = 50,000 central portion of the associated heavy chain is preferentially unfolded. This unfolding is accompanied by the loss in ATPase function where the rate of inactivation can be correlated with the loss in the amount of the Mr = 50,000 fragment generated under standard tryptic digestion conditions. The residual protein appears to be a soluble aggregate of a complex of the Mr = 27,000, 21,000, and light chain with no intact Mr = 50,000 fragment. Tryptic digestions in the presence of MgATP are restricted to the usual linker regions and the Mr = 50,000 fragment is completely protected from attack. Binding of actin to subfragment 1 also results in the protection of the Mr = 50,000 segment and of the Mr = 50,000/21,000 junction from tryptic attack. The data suggest that, in terms of methanolic perturbation, the subfragment 1 appears to be comprised of two domains with differential stability. One domain appears to be the central Mr = 50,000 segment of the heavy chain which is preferentially unfolded by methanol and requires the presence of MgATP or of actin for stabilization. The other domain is more stable and appears to consist of the interacting Mr = 27,000, 21,000, and light chain. The results also suggest that the integrity of the Mr = 50,000 segment is essential for the ATPase function of the protein.

Isolation of heavy chain isoenzymes of myosin subfragment 1 by high performance ion exchange chromatography.

The procedure of high performance ion-exchange chromatography has been used to fractionate subfragment 1 of myosin (SF1) into its isoenzymic forms. In contrast to conventional ion-exchange procedures which yield two fractions corresponding to SF1(A1) and SF1(A2), the high performance liquid chromatography (HPLC) procedure resolves SF1 into four discrete fractions. The first pair that is eluted appears to be A1-containing isoenzymes while the latter pair corresponds to the A2 forms based on their polypeptide compositions by gel electrophoresis in the presence of sodium dodecyl sulfate. By gel electrophoresis under nondenaturing conditions it is not possible to differentiate between the two fractions corresponding to each isoenzyme. Although very minor differences between the fractions can be seen by the presence of extraneous peptides, these are present in far below stoichiometric amounts and, therefore, make it very unlikely that the superior fractionation by the HPLC procedure is based on their presence. An examination of the heavy chain heterogeneity in each of these fractions by peptide mapping revealed that the extra separation was based on this factor. Thus the HPLC procedure is capable of providing separation of SF1 into heavy chain-based isozymes as well as the light chain forms. ATPase measurements of these fractions reveal only minor differences in the Ca2+- and EDTA-activated ATPase.

The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity.

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (A1) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under nondenaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 degrees C or 37 degrees C with free A1 or A2 to form the subfragment 1 isozymes SF1(A1) or SF1(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding domain.

Subunit interactions in myosin subfragment 1. Subunit scrambling is independent of catalytic center involvement.

Evidence is presented that, under conditions of 4.7 M NH4Cl and 10 mM Mg-ATP where no subunit dissociation can be detected by transport methods, a dynamic equilibrium exists in subfragment 1 between the associated and dissociated subunits. This is readily discerned by the formation of hybrid subfragment 1 species when a subfragment 1 isozyme is incubated with excess free light chains of the alternate isozyme. A similar process occurs with p-N,N'-phenylenedimaleimide (pPDM)-modified subfragment 1 containing [14C]Mg-ADP, but in this case, although extensive amounts of hybrid are formed, no loss of the trapped nucleotide is observed. Subunit scrambling without loss of the trapped nucleotide is apparent from incubating pPDM-SF1(A2)-[14C]Mg-ADP with unmodified SF1(A1) under similar conditions since the mixture subsequently contains SF1(A1), SF1(A2)h, pPDM-SF1(A1)h-[14C]Mg-ADP and pPDM-SF1(A2)-[14C]Mg-ADP. These data show that the nucleotide trapped in the presumptive active site does not escape during the dissociation-reassociation cycle, and suggest that the ATPase site resides solely on the heavy chain.

Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Formation and properties of thermal hybrids.

The formation of hybrid myosin and subfragment 1 species by incubation of these proteins with free alkali light chains at physiological ionic and temperature conditions is described. Exchange of bound alkali light chain on myosin by free alkali light chains under these conditions is readily demonstrated from the subunit composition of the isolated myosin. Therefore, the light chain exchange previously described for the one-headed subfragment 1 [Sivaramakrishnan, M., & Burke, M (1981) J. Biol. Chem. 256, 2607--2610] also occurs in the two-headed myosin molecule. It is found than the isozyme to hybrid transformation is dependent on both the temperature and the ionic strength of the incubation mixture but is relatively independent of pH in the range 6.5--8.0. A comparison of the SF1(A1) leads to SF1(A2)h system with the SF1(A2) leads to SF1(A1)h system indicates that more hybrid is formed in the latter case. With the assumption that hybrid formation reflects the degree of reversible dissociation exhibited by the isozyme, under the particular experimental condition employed, the data signify that the subunit interactions in the two isozymes are not identical and that the heavy chain--A1 interactions are significantly more stable that the heavy chain--A2 ones. An examination of the ATPase properties of the thermal hybrids in the presence and absence of actin indicates close similarities to their corresponding "native" isozymic counterparts.

Studies on the interaction of myosin subfragment 1 and immobilized nucleotide. Evidence for different binding domains operating under different solvent conditions.

The binding interaction between myosin subfragment 1 isozymes with immobilized nucleotide, where they show differential behavior, has been examined. By employing subfragment 1 hybrids formed by crosses between heavy and alkali light chains, it is possible to demonstrate that the differential behavior is modulated by the alkali light chain component of the protein and not by differences in the heavy chain subunits in these isozymes resulting from the proteolytic treatment used in their formation. The fact that the free alkali light chains show weak differential binding under these conditions suggests that the binding in the case of the subfragment 1 isozymes may occur at a site distinct from the ATPase site. This was substantiated by examining the behavior of subfragment 1 containing [14C] MgADP noncovalently trapped in the ATPase site by the bifunctional reagent N, N-p-phenylenedimaleimide, on agarose-ATP. The data suggest that different ATP binding domains may be operating in myosin depending on the ionic conditions being employed.

Studies on the subunit interactions of skeletal muscle myosin subfragment 1. Evidence for subunit exchange between isozymes under physiological ionic strength and temperature.

Evidence is presented that under physiological conditions of ionic strength and temperature, where myosin Subfragment 1 is hydrolyzing MgATP, the interaction between its subunits is extremely labile. Incubation of [3H]N-ethylmaleimide-SF1(A1) with N-ethylmaleimide-SF1(A2) in the presence of 10 mM MgATP at 37 degrees C resulted in the exchange of subunits between these isozymes. This is readily discernible from the subunit composition and distribution of the 3H label after separation of the isozymes by ion exchange chromatography. Moreover, incubation of unmodified SF1(A1) or SF1(A2) with the free Alkali light chains A2 and A1, respectively, under the same conditions led to the formation of significant amounts of the hybrid species. These findings suggest that in vivo the Alkali light chain-heavy chain interaction of Subfragment 1 is in a state of dynamic equilibrium between associated and dissociated states.