Specific, Reversible Cytostatic Protection of Normal Cells Against Negative Effects of Chemotherapy

Chemotherapy is a common and effective method to treat many forms of cancer. However, treatment of cancer with chemotherapy has severe side effects which often limit the doses of therapy administered. Because some cancer chemotherapeutics target proliferating cells and tissues, all dividing cells, whether normal or tumor, are affected. Cell culture studies have demonstrated that UCN-01 is able to reversibly and selectively arrest normal dividing cells; tumor cells lines do not undergo this temporary arrest. Following UCN-01 treatment, normal cells displayed a 50-fold increase in IC50 for camptothecin; tumor cells showed no such increased tolerance. We have examined the response of the proliferating tissues of the mouse to UCN- 01 treatment, using the small bowel epithelium as a model system. Our results indicate that UCN-01 treatment can cause a cell cycle arrest in the gut epithelium, beginning 24 hours following UCN-01 administration, with cell proliferation remaining suppressed for one week. Two weeks post-UCN-01 treatment the rate of proliferation returns to normal levels. 5-FU administered during this period demonstrates that UCN-01 is able to provide protection to normal cells of the mouse within a narrow window of efficacy, from three to five days post-UCN-01. UCN-01 pretreated mice displayed improved survival, weight status and blood markers following 5-FU compared to control mice, indicating that UCN-01 can protect normal dividing tissues. The mechanism by which UCN-01 arrests normal cells in vivo was also examined. We have demonstrated that UCN-01 treatment in mice causes an increase in the G1 phase cell cycle proteins cdk4 and cyclin D, as well as the inhibitor p27. Phosphorylated Rb was also elevated in the arrested cells. These results are a departure from cell culture studies, in which inhibition of G1 phase cyclin dependent kinases led to hyposphosphorylation of Rb. Future investigation will be required to understand the mechanism of UCN-01 action. This is important information, especially for identification of alternate compounds which could provide the protection afforded by UCN-01.

Spike timing dependent plasticity: a consequence of more fundamental learning rules.

Spike timing dependent plasticity (STDP) is a phenomenon in which the precise timing of spikes affects the sign and magnitude of changes in synaptic strength. STDP is often interpreted as the comprehensive learning rule for a synapse - the "first law" of synaptic plasticity. This interpretation is made explicit in theoretical models in which the total plasticity produced by complex spike patterns results from a superposition of the effects of all spike pairs. Although such models are appealing for their simplicity, they can fail dramatically. For example, the measured single-spike learning rule between hippocampal CA3 and CA1 pyramidal neurons does not predict the existence of long-term potentiation one of the best-known forms of synaptic plasticity. Layers of complexity have been added to the basic STDP model to repair predictive failures, but they have been outstripped by experimental data. We propose an alternate first law: neural activity triggers changes in key biochemical intermediates, which act as a more direct trigger of plasticity mechanisms. One particularly successful model uses intracellular calcium as the intermediate and can account for many observed properties of bidirectional plasticity. In this formulation, STDP is not itself the basis for explaining other forms of plasticity, but is instead a consequence of changes in the biochemical intermediate, calcium. Eventually a mechanism-based framework for learning rules should include other messengers, discrete change at individual synapses, spread of plasticity among neighboring synapses, and priming of hidden processes that change a synapse's susceptibility to future change. Mechanism-based models provide a rich framework for the computational representation of synaptic plasticity.

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

STABILITY AND FOLDING OF MUTANT FORMS OF ISO-1 CYTOCHROME-C FROM SACCHAROMYCES CEREVISIAE

Partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae were obtained by replacements of the evolutionarily conserved proline 71 with valine, isoleucine and threonine (Ernst et.al.,1985). Pro-71 lies at the juncture of two short helical regions and is believed to be important for proper local polypeptide chain folding within the iso-1-cytochrome c structure.^ To study folding in the absence of intermolecular disulfide dimer formation the free sulfhydryl group of Cys-102 was modified in both wild type and mutant proteins with an alkylating reagent, methyl methanethiosulfonate. Spectral analysis of the wild type and mutant proteins shows that the native-like functional (or partially functional) folded structure of cytochrome c is retained in the chemically modified derivatives. The replacement of Pro-71 with valine, isoleucine or threonine reduces the intensity of the 696 nm absorbance band which is an indicator of the Met-80 ligation to the heme. Thermal stability and guanidine hydrochloride unfolding studies of the mutant proteins shows a destabilization of the protein as a result of mutation. The degree of destabilization depends on the chemical nature of the substituent amino acid in the mutant protiens.^ Kinetics of folding/unfolding reactions of the proteins were monitored by fluorescence changes using stopped flow mixing to obtain guanidine hydrochloride concentration jumps ending below, within, and above the transition zone. The replacement of Pro-71 alters the rate on one of the fastest phases, $\tau\sb3$, while the two other phases, $\tau\sb1$ & $\tau\sb2$, remain the same.^ Slow refolding kinetic studies indicate that replacement of Pro-71 does not completely eliminate the absorbance or fluorescence detected slow phases leading to the conclusion that Pro-71 is not involved in the generation of the slow phases in the folding kinetics of iso-1-cytochrome c.^ The alkaline conformational change involving the disappearance of the 696 nm absorbance band occurs with increasing pH in the alkaline pH region (Davis et al., 1974). The apparent pK of this conformational change in mutant proteins is shifted as much as two pH units compared to wild type. The equilibrium and kinetic data of alkaline transition for the wild type follows a simple mechanism proposed by Davis et al., (1974) for horse heart cytochrome c. A more complex mechanism is proposed for the behavior of the mutant proteins. ^

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^

A comparative review of water disinfection methods appropriate for developing countries and their efficacy, cost-efficiency, and usability

Developing countries are heavily burdened by limited access to safe drinking water and subsequent water-related diseases. Numerous water treatment interventions combat this public health crisis, encompassing both traditional and less-common methods. Of these, water disinfection serves as an important means to provide safe drinking water. Existing literature discusses a wide range of traditional treatment options and encourages the use of multi-barrier approaches including coagulation-flocculation, filtration, and disinfection. Most sources do not delve into approaches specifically appropriate for developing countries, nor do they exclusively examine water disinfection methods.^ The objective of this review is to focus on an extensive range of chemical, physio-chemical, and physical water disinfection techniques to provide a compilation, description and evaluation of options available. Such an objective provides further understanding and knowledge to better inform water treatment interventions and explores alternate means of water disinfection appropriate for developing countries. Appropriateness for developing countries corresponds to the effectiveness of an available, easy to use disinfection technique at providing safe drinking water at a low cost.^ Among chemical disinfectants, SWS sodium hypochlorite solution is preferred over sodium hypochlorite bleach due to consistent concentrations. Tablet forms are highly recommended chemical disinfectants because they are effective and very easy to use, but also because they are stable. Examples include sodium dichloroisocyanurate, calcium hypochlorite, and chlorine dioxide, which vary in cost depending on location and availability. Among physio-chemical disinfection options, electrolysis which produces mixed oxidants (MIOX) provides a highly effective disinfection option with a higher upfront cost but very low cost over the long term. Among physical disinfection options, solar disinfection (SODIS) applications are effective, but they treat only a fixed volume of water at a time. They come with higher initial costs but very low on-going costs. Additional effective disinfection techniques may be suitable depending on the location, availability and cost.^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^