Kinetic Parameters of Thymidine Kinase and DNA Synthesis During Liver Regeneration: Role Ofthyroid Hormones

The role of thyroid hormones in DNA synthesis and in the activity of Thymidille kinase (TK), a key regulatory enzyme of DNA synthesis was studied in proliferating hepatocytes in vivo. Liver regeneration after partial hepatectomy was used as a model for controlled cell division in rats having different thyroid status - euthyroid, hypothyroid and 3,3',5'-triiodo-L-thyronine (T))-heated hypothyroid. Partial hepatectomy caused a significant elevation of DNA synthesis (p<0.01) in all the three groups compared to their sham-operated counterparts. Hypothyroid liepatectomised animals showed significantly lower (p<0.01) level of DNA synthesis than euthyroid hepatectomised animals. A single subcutaneous close of 1'3 to hypothyroid shamoperated animals resulted in a significant increase (p<0.01) of DNA synthesis in the intact liver. 17tis was comparable to the level of DNA synthesis occurring in regenerating liver of euthyroid animals. In hypothyroid hepatectomised animals, "1'3 showed an additive effect on l)NA synthesis and this group exhibited maximum level of DNA synthesis (p<0.0I ). Studies of the kinetic parameters of TK show that the Michelis-Menten constant, (K111) of TK for thymidine was altered by the thyroid status. K11 increased significantly (p<0.01) in untreated hypothyroid animals when compared to the euthyroid rats. '13 treatment of hypothyroid animals reversed this effect and this group showed the lowest value for K111 (p<0.01). Thus our results indicate that thyroid hormones can influence DNA synthesis during liver regeneration and they may regulate the activity of enzymes such as 17rymidine kinase which are important for DNA synthesis and hence cell division.

Effect Insulin on DNA Synthesis and Kinetic Parameters of Thymidine Kinase During Liver Regenaration

The effect of insulin on cell proliferation in vivo has been studied in hepatectomised streptozotocin- diabetic rats. The extent of cell proliferation in sham and hepatectomized- control, diabetic and insulin treated rats were monitored by determining DNA content and [3H]thymidine incorporation into DNA. The kinetic parameters of thymidine kinase a regulatory enzyme for DNA synthesis was also studied in these groups. The rate of DNA synthesis in liver of streptozotocin -diabetic rats was significantly higher 24 hrs post-hepatectomy compared to control and insulin treated diabetic groups. Kinetic studies of thymidine kinase revealed that there was no change in the Michaelis -Menten constant (Km) whereas maximum velocity (Vmax) was elevated in the diabetic hepatectomized groups compared to control and insulin treated hepatectomized groups. Thus our study elucidates the role of insulin in thymidine kinase activity and DNA synthesis.

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme.Km, Vmax, and Kcat of the enzyme were 4.727 9 10-2 mg/ml, 394.68 U, and 4.2175 9 10-2 s-1, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65 C), withmaximumactivity at pH 11 and 60 C. CaCl2 (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65 C. Hg2?, Cu2?, Fe3?, Zn2?, Cd?, and Al3? inhibited enzyme activity, while 1 mMCo2? enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.

Provenance, Isolation and Characterisation of Organic Matter in the Cochin Estuarine Sediment-“a Diagenetic Amino Acid Marker Scenario

Cochin estuarine system is among the most productive aquatic environment along the Southwest coast of India, exhibits unique ecological features and possess greater socioeconomic relevance. Serious investigations carried out during the past decades on the hydro biogeochemical variables pointed out variations in the health and ecological functioning of this ecosystem. Characterisation of organic matter in the estuary has been attempted in many investigations. But detailed studies covering the degradation state of organic matter using molecular level approach is not attempted. The thesis entitled Provenance, Isolation and Characterisation of Organic Matter in the Cochin Estuarine Sediment-“ A Diagenetic Amino Acid Marker Scenario” is an integrated approach to evaluate the source, quantity, quality, and degradation state of the organic matter in the surface sediments of Cochin estuarine system with the combined application of bulk and molecular level tools. Sediment and water samples from nine stations situated at Cochin estuary were collected in five seasonal sampling campaigns, for the biogeochemical assessment and their distribution pattern of sedimentary organic matter. The sampling seasons were described and abbreviated as follows: April- 2009 (pre monsoon: PRM09), August-2009 (monsoon: MON09), January-2010 (post monsoon: POM09), April-2010 (pre monsoon: PRM10) and September- 2012 (monsoon: MON12). In order to evaluate the general environmental conditions of the estuary, water samples were analysed for water quality parameters, chlorophyll pigments and nutrients by standard methods. Investigations suggested the fact that hydrographical variables and nutrients in Cochin estuary supports diverse species of flora and fauna. Moreover the sedimentary variables such as pH, Eh, texture, TOC, fractions of nitrogen and phosphorous were determined to assess the general geochemical setting as well as redox status. The periodically fluctuating oxic/ anoxic conditions and texture serve as the most significant variables controlling other variables of the aquatic environment. The organic matter in estuary comprise of a complex mixture of autochthonous as well as allochthonous materials. Autochthonous input is limited or enhanced by the nutrient elements like N and P (in their various fractions), used as a tool to evaluate their bioavailability. Bulk parameter approach like biochemical composition, stoichiometric elemental ratios and stable carbon isotope ratio was also employed to assess the quality and quantity of sedimentary organic matter in the study area. Molecular level charactersation of free sugars and amino acids were carried out by liquid chromatographic techniques. Carbohydrates are the products of primary production and their occurrence in sediments as free sugars can provide information on the estuarine productivity. Amino acid biogeochemistry provided implications on the system productivity, nature of organic matter as well as degradation status of the sedimentary organic matter in the study area. The predominance of carbohydrates over protein indicated faster mineralisation of proteinaceous organic matter in sediments and the estuary behaves as a detrital trap for the accumulation of aged organic matter. The higher lipid content and LPD/CHO ratio pointed towards the better food quality that supports benthic fauna and better accumulation of lipid compounds in the sedimentary environment. Allochthonous addition of carbohydrates via terrestrial run off was responsible for the lower PRT/CHO ratio estimated in thesediments and the lower ratios also denoted a detrital heterotrophic environment. Biopolymeric carbon and the algal contribution to BPC provided important information on the better understanding the trophic state of the estuarine system and the higher values of chlorophyll-a to phaeophytin ratio indicated deposition of phytoplankton to sediment at a rapid rate. The estimated TOC/TN ratios implied the combined input of both terrestrial and autochthonous organic matter to sedimentsAmong the free sugars, depleted levels of glucose in sediments in most of the stations and abundance of mannose at station S5 was observed during the present investigation. Among aldohexoses, concentration of galactose was found to be higher in most of the stationsRelative abundance of AAs in the estuarine sediments based on seasons followed the trend: PRM09-Leucine > Phenylalanine > Argine > Lysine, MON09-Lysine > Aspartic acid > Histidine > Tyrosine > Phenylalanine, POM09-Lysine > Histadine > Phenyalanine > Leucine > Methionine > Serine > Proline > Aspartic acid, PRM10-Valine > Aspartic acid > Histidine > Phenylalanine > Serine > Proline, MON12-Lysine > Phenylalanine > Aspartic acid > Histidine > Valine > Tyrsine > MethionineThe classification of study area into three zones based on salinity was employed in the present study for the sake of simplicity and generalized interpretations. The distribution of AAs in the three zones followed the trend: Fresh water zone (S1, S2):- Phenylalanine > Lysine > Aspartic acid > Methionine > Valine ῀ Leucine > Proline > Histidine > Glycine > Serine > Glutamic acid > Tyrosine > Arginine > Alanine > Threonine > Cysteine > Isoleucine. Estuarine zone (S3, S4, S5, S6):- Lysine > Aspartic acid > Phenylalanine > Leucine > Valine > Histidine > Methionine > Tyrosine > Serine > Glutamic acid > Proline > Glycine > Arginine > Alanine > Isoleucine > Cysteine > Threonine. Riverine /Industrial zone (S7, S8, S9):- Phenylalanine > Lysine > Aspartic acid > Histidine > Serine > Arginine > Tyrosine > Leucine > Methionine > Glutamic acid > Alanine > Glycine > Cysteine > Proline > Isoleucine > Threonine > Valine. The abundance of AAs like glutamic acid, aspartic acid, isoleucine, valine, tyrosine, and phenylalanine in sediments of the study area indicated freshly derived organic matter.