Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

The kinetics of germination in bacterial spores

The entire absorbance vs. time curve of a sample of germinating bacterial spores can be accurately described by a model which considers that the spores rate of entry into the phase initiation of germination is determined by transitions between three spore states. The first of these transitions is easily identified with the triggering event, while the existence of the intermediate state, and its identification with the triggered spore, can be established from theoretical as well as experimental considerations. The observed sample lag time is seen to arise from the position of the measured event in the single spore in the sequence of indices of germination. Consideration that the single spore may effect the measured change in a complex way over a finite interval of time leads to a mathematical formulation of our model which can describe the germination process whatever the endpoint chosen for its observation.     

Structural basis for the dual thymidine and thymidylate kinase activity of herpes thymidine kinases

Crystal structures of equine herpesvirus type-4 thymidine kinase (EHV4-TK) in complex with (i). thymidine and ADP, (ii). thymidine and SO(4) and the bisubstrate analogs, (iii). TP(4)A, and (iv). TP(5)A have been solved. Additionally, the structure of herpes simplex virus type-1 thymidine kinase (HSV1-TK) in complex with TP(5)A has been determined. These are the first structures of nucleoside kinases revealing conformational transitions upon binding of bisubstrate analogs. The structural basis for the dual thymidine and thymidylate kinase activity of these TKs is elucidated. While the active sites of HSV1-TK and EHV4-TK resemble one another, notable differences are observed in the Lid regions and in the way the enzymes bind the base of the phosphoryl-acceptor. The latter difference could partly explain the higher activity of EHV4-TK toward the prodrug ganciclovir.     

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5′ exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R² values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.     

Temperature-pH effect upon germination of bacterial spores.

Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures. The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C. A possible mechanism involved in this phenomenon is discussed.     

Close relation between fetal thymidine kinase and thymidylate kinase activities in breast cancer

In human breast cancers, assays on thymidine kinase activity revealed the synthesis of large amounts of d-TTP. This fact suggested the presence of thymidylate kinase closely associated with thymidine kinase. Results obtained with experimental tumors were quite different. These tumors appeared inadequate for the study on thymidine metabolism in mammary cancers.     

Enzyme regulation during the synchronous nuclear cycle of Physarum polycephalum: Effects of methotrexate and hydroxyurea on thymidine kinase and deoxycytidine kinase

Specific enzyme activities of thymidine kinase (TK) and deoxy-cytidine kinase (dCK) increase sharply at the onset of synchronous mitosis in macroplasmodia of Physarum polycephalum. They reach a maximum in early S-phase (Physarum lacks a G1 period) and decline to a minimum in late G2. Partial inhibition of DNA synthesis with methotrexate (MTX) or hydroxyurea (HU) retards the onset of the next mitosis and provokes a superinduction of both enzymes, with dCK responding stronger than TK. The temporal pattern observed suggests that the drugs interfere with the postmitotic down-regulation of enzyme expression, possibly due to alterations of the chromatin structure. Moderate inhibition of DNA synthesis still permits the appearance of (delayed) mitoses associated with peaks of enzyme activity at elevated levels. On the other hand, stronger inhibition completely suppresses the onset of mitosis and keeps the enzyme activities at an elevated level without further oscillations. The timing mechanism of periodic enzyme induction therefore appears to be functionally linked to the mitotic signal and does not persist under a stringent DNA block.     

Chemical germination of native and cation-exchanged bacterial spores with trifluoperazine

The calmodulin antagonist trifluoperazine and its analog chlorpromazine, both amphipaths, induced chemical germination of spores of various species, as do many surfactants. Cation load can greatly influence this response. Calmodulin antagonism does not seem to be involved. A new fluorometric assay for dipicolinic acid based on the fluorescence of the dipicolinic acid chelate of Tb3+ was found to be simple and sensitive.     

Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor

Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.     

Increase in liver and kidney deoxycytidine kinase activity linked to neoplastic transformation

Deoxycytidine kinase activity in normal rat liver cytosol was low (0.8 nmol/hr/mg protein); it increased 2–26-fold in 12 lines of chemically-induced, transplantable rat hepatomas of different growth rates. The increased kinase activity correlated positively with the hepatoma growth rate. The kinase activity did not change in the regenerating liver and the activity in the differentiating, neonatal rat liver was similar to values in adult liver. Deoxycytidine kinase activity in 2 chemically-induced, transplantable rat kidney tumors was increased to twice the value found in normal renal cortex. Among 15 normal rat tissues examined the highest kinase activities were observed in thymus, bone marrow and spleen. Of the normal and malignant rat tissues tested, only testis had detectable cytidine deaminase activity.     

Database searching for thymidine and thymidylate kinase inhibitors using three-dimensional structure-based methods

Structure-based drug design methods were used to search for novel inhibitors of herpes simplex virus type 1 (HSV-1) thymidine kinase and Mycobacterium tuberculosis thymidylate kinase. The method involved the use of crystal structure complexes to guide database searching for potential inhibitors. A number of weak inhibitors of HSV-2 were identified, one of which was found to inhibit HSV-1 TK and HSV-1 TK-deficient viral strains. Each compound tested against M. tuberculosis thymidylate kinase was found to have some activity. The best of these compounds was only 4.6-fold less potent than 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP). This study demonstrates the utility of structure-based drug design methods in the search for novel enzyme inhibitors.     

Chemical germination of native and cation-exchanged bacterial spores with trifluoperazine.

The calmodulin antagonist trifluoperazine and its analog chlorpromazine, both amphipaths, induced chemical germination of spores of various species, as do many surfactants. Cation load can greatly influence this response. Calmodulin antagonism does not seem to be involved. A new fluorometric assay for dipicolinic acid based on the fluorescence of the dipicolinic acid chelate of Tb3+ was found to be simple and sensitive.