Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus-induced expression of CKIs p16(ink4), p21(waf/cip1), and p27(kip1), as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation- and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1-associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.

Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis.

Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentrations of KCl, but the underlying molecular mechanisms are not yet clear. Although caspases have been postulated to be involved in CGN cell death, inhibitors of caspases failed to prevent apoptosis under our culture conditions, suggesting an involvement of other molecules and pathways. We find that inhibitors of cyclin-dependent kinases--flavopiridol, olomoucine, and roscovitine--protect CGNs from KCl withdrawal-induced apoptosis, suggesting that cell cycle components play a significant role in the death of these neurons. Analysis of the different cell cycle regulatory elements in this model revealed that apoptosis is preceded by an increase in the level of cyclin E protein, with elevated nuclear levels of cyclin D1 and with enhanced activity of the cyclin D1- and E- associated kinases. In addition, there was a significant decrease in the level of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these changes, analysis of a major substrate of cyclin-activated cdks, retinoblastoma protein (Rb), showed an increase in the level of phosphorylated forms within 1 hr of KCl withdrawal. Moreover, the overall levels of Rb protein were significantly reduced within 6-12 hr of KCl withdrawal and did so by a caspase-independent mechanism. All of these responses were blocked by cdk inhibitors. These findings indicate that cdks act at an early step in the pathway by which KCl withdrawal induces apoptotic death of cerebellar granule cells and suggest that additional elements of the cell cycle machinery participate in this mechanism.

Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage.

Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.

Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation, and superoxide production.

The effects of pentoxifylline (Trental) on human neutrophil CR3 up-modulation, degranulation, and superoxide production were studied. We used the chemotactic peptide fMLP and the phorbol ester PMA as soluble stimuli, and beta-glucan particles as a CR3-specific solid phase stimulus of neutrophil superoxide production. Since neutrophils have adenosine A2 receptors, we compared effects of pentoxifylline to effects of adenosine, and we also looked at the effect of cytochalasin B, which breaks up actin filaments. Pentoxifylline inhibited both CR3 up-modulation and degranulation of myeloperoxidase and lysozyme. Pentoxifylline is a more potent inhibitor of fMLP- compared to PMA-induced degranulation, and is especially potent against superoxide production. While pentoxifylline is less potent than adenosine in its inhibition of fMLP-induced superoxide production, it is more potent in its inhibition of PMA- and beta-glucan particle-stimulated superoxide production. Cytochalasin B, which enhances degranulation and fMLP-stimulated superoxide production, was found to inhibit beta-glucan particle-stimulated superoxide production. These findings are consistent with the hypothesis that pentoxifylline can affect both the cytoskeletal architecture of unstimulated neutrophils and the activation and responses of neutrophils which involve actin polymerization and receptor-cytoskeletal interactions.

Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor.

Dichlorofluorescein (DCFH) oxidation assay measures hydrogen peroxide (H2O2), which is a derivative of superoxide anion. We found that a calmodulin antagonist, W-13, which is known to inhibit superoxide anion generation enhanced the capacity of human neutrophils to oxidize DCFH. To investigate this discrepancy we studied the role of nitric oxide (NO) in DCFH oxidation. Pure NO was capable of oxidizing DCFH, and the product formed had spectral properties identical to oxidized DCFH produced by H2O2. The arginine analog, NG-monomethyl-L-arginine (NMMA), which inhibits NO production, in combination with W-13 completely inhibited the stimulus-induced increase in DCFH oxidation. We conclude that the oxidation of DCFH in human neutrophils can occur by either H2O2 or NO.

Lymphoid organ production of immunomodulatory eicosanoids in mice resistant to neonatal tolerance induction.

Neonatally induced tolerance of class I and class II alloantigens is difficult to achieve in certain I-E non-expressing hosts that received semiallogeneic cells at birth from strains of mice that express I-E molecules. Although clonal deletion occurs ubiquitously after infusion of the tolerogen-bearing inoculum, the majority of these mice ultimately regain the capacity to reject donor-specific skin graft challenges in adulthood and this is associated with a reacquisition of I-E recognizing and alloreactive T cells as well as a loss of donor chimeric cells. In this study, we determined whether production levels of the eicosanoids prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), both potent modifiers of lymphocyte function, were altered in lymphoid organs concomitant with a breakdown of tolerance in these mice. The levels of TxB2 and PGE2 produced by lymphoid organs were measured in the early/late post-partum periods and immediately before and after skin grafting in B10.S mice (H-2s/I-E-) that had been injected at birth with (B10.S x B10.A)F1 (H-2k/d, I-E+) lymphohematopoietic cells. Phenotypic (e.g., %V beta 11+ T cells) and functional parameters of host donor-reactive effector cell populations along with chimerism were determined simultaneously. We found that TxB2 and PGE2 production fluctuated in the early postnatal periods in naive mice and that the neonatally injected counterparts showed a significant alteration from this pattern, particularly with PGE2. As adults, injected hosts maintained an altered pattern of eicosanoid metabolism and this was accentuated after the rejection or acceptance of a donor-specific skin allograft. Specific patterns emerged after transplant challenge such that neonatally injected mice deleted of V beta 11+ T cells before grafting differed in their eicosanoid secretory profiles; moreover, injected mice that accepted (i.e., tolerant) the donor-specific allograft had a markedly different TxB2 and PGE2 profile than injected/rejecting hosts. In naive mice, the application of 2 subsequent grafts elicited a release of splenic TxB2 and PGE2 that mimicked the pattern seen in the neonatally injected hosts after 1 graft--these latter results give preliminary indication that the generation of memory T cells and the re-exposure to specific alloantigen coincides with a derangement in eicosanoid metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of a vitronectin binding region of plasminogen activator inhibitor-1.

The PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70, 1-105, 1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

The effects of octreotide on GH receptor and IGF-I expression in the GH-deficient rat.

Somatostatin has been suggested to influence the somatotrophic axis outside the central nervous system, in reducing GH-induced IGF-I mRNA and IGF-I generation. This study aimed to determine whether such effects were mediated via the GH receptor (GHR). GH-deficient dwarf rats aged 45-47 days (n = 8 per group) received twice daily subcutaneous injections of octreotide (1 mg/kg) (group O), saline (group S), octreotide (1 mg/kg) plus bovine GH (0.25 mg/kg) (group OG), or bovine GH (0.25 mg/kg) plus saline (group G) for 10 days. Octreotide-treated animals had less weight gain compared with saline-treated animals, but not when GH cotreated (group OG vs G). Octreotide had an overall effect on decreasing length gain (P < 0.01). Serum IGF-I (ng/ml) was reduced by octreotide (group O 171 +/- 11, group S 239 +/- 20, P < 0.01; group OG 283 +/- 30, group G 362 +/- 10, P < 0.001), as was serum insulin (P < 0.001). A significant decrease in hepatic and muscle IGF-I mRNA expression was found as expected, yet this was not associated with decreased hepatic GHR expression. Rather, an increase in hepatic 125I-bovine GH specific binding was observed (P < 0.001) and, in GH-cotreated animals (OG), hepatic GHR and GH binding protein (GHBP) mRNA expression were also increased by octreotide by approximately 40%. In muscle, octreotide was associated with an approximately 30% decrease in GHBP mRNA and no effect on GHR mRNA. This study suggests that the suppressive effects of octreotide on IGF-I metabolism, at least in liver, are not mediated via down-regulation of GHR expression, but more likely by direct effects on IGF-I expression.

Differential patterns of T cell clonal deletion in neonatal H-2 tolerance and I-E/Mls induced self-tolerance.

The pattern of clonal deletion of putative I-E-reactive (V beta 11) and Mls-reactive (V beta 3) T cells was evaluated and compared by cytofluorographic and immunohistochemical methods in a model of neonatal H-2 tolerance and in I-E- or Mls-bearing strains of mice which normally delete these cell populations (self-tolerance). The ontogeny of deletion of V beta 11+ cells was studied by evaluating thymic changes from birth until maturity in B10.S (H-2s/I-E-), B10.A (H-2k/d/I-E+) and B10.S mice intravenously infused at birth with (B10.SxB10.A)F1 lymphohaematopoietic cells. The reduction in V beta 11+ cells was most prevalent in the medullary region of the naive B10.A and neonatally injected B10.S animals and was corroborated by flow cytometry which demonstrated a marked reduction in single CD4 and CD8 positive B beta 11 T cells when compared to naive B10.S mice. However, immunohistochemistry illustrated that 'deletion' was never complete since V beta 11+ cells remained in the thymic cortex and splenic lymphoid follicles. By comparison, DBA/2 mice (Mlsc+ and previously documented to have decreased levels of V beta 3+ cells) showed a different pattern of deletion of V beta 3+ T cells than what was found for T cells bearing V beta 11 in animals deleting this population. DBA/2 thymi contained fewer thymic V beta 3- cells and there was more complete elimination of these cells, particularly in the periphery, by flow cytometry and immunohistology. The mice which do not delete V beta 3 cells (Mlsc-) showed that the majority of V beta 3- cells were located in the medulla with a few cells distributed in the cortical region. This pattern was notably different than the distribution of V beta 11 cells in thymi. Despite their location by histology, the majority of remaining V beta 3+ cells were dual CD4/CD8 positive (CD4+CD8+) by flow cytometric analysis. Our data illustrate that V beta 11 and V beta 3 T cells appear to be eliminated (i.e. 'deleted') at similar stages of maturation (single positive) during self-tolerance as well as in a neonatal H-2 tolerance model. However, the degree of elimination and the location of the cells remaining in these mice is dramatically different, depending on which T cell population is being evaluated and which deleting ligand is presented intrathymically. Thus, the accepted tenet of dual CD4+CD8+ cells localizing to the thymic cortex appears to have exceptions.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophilicity-based charge transfer descriptor.

In line with the charge transfer (DeltaNmax = -mu/eta) proposed by Parr et al. (Parr, R. G.; Szentpály, L. V.; Liu, S. J. Am. Chem. Soc. 1999, 121, 1922), we propose an electrophilicity-based charge transfer (ECT) descriptor in this paper and validate it through the interaction between a series of chlorophenols and DNA bases. Application of ECT can be extended to the interaction of any toxin with the biosystem.

Does rectus sheath infusion of bupivacaine reduce postoperative opioid requirement?

INTRODUCTION: The aim of this work was to assess the effect of intermittent bupivacaine infusion into rectus sheath space on postoperative opioid requirement, postoperative pain score and peak expiratory flow rate. PATIENTS AND METHODS: A prospective, randomised study involving patients undergoing midline laparotomy. Patients were randomised to receive either intermittent infusion of bupivacaine 0.25% or normal saline via catheters placed in the rectus sheath for 48 h after operation. All patients received intravenous morphine infusion on demand with a patient-controlled analgesic device (PCAD). RESULTS: Forty ASA I-III patients were studied. Nineteen were randomised to receive bupivacaine and 21 patients received normal saline. Patient characteristics and surgical variables were comparable in the two groups. The mean wound lengths were similar. There was no statistically significant difference in postoperative opioid requirement, postoperative pain score and peak expiratory flow rate between the two groups. CONCLUSIONS: Intermittent bupivacaine infusion into the rectus sheath space after midline laparotomy does not reduce postoperative opioid requirement nor does it affect postoperative pain score or peak expiratory flow rate.

Multiphilic descriptor for chemical reactivity and selectivity.

In line with the local philicity concept proposed by Chattaraj et al. (Chattaraj, P. K.; Maiti, B.; Sarkar, U. J. Phys. Chem. A. 2003, 107, 4973) and a dual descriptor derived by Morell, Grand and Toro-Labbé, (J. Phys. Chem. A 2005, 109, 205), we propose a multiphilic descriptor. It is defined as the difference between nucleophilic (omega(k)+) and electrophilic (omega(k)-) condensed philicity functions. This descriptor is capable of simultaneously explaining the nucleophilicity and electrophilicity of the given atomic sites in the molecule. Variation of these quantities along the path of a soft reaction is also analyzed. Predictive ability of this descriptor has been successfully tested on the selected systems and reactions. Corresponding force profiles are also analyzed in some representative cases. Also, to study the intra- and intermolecular reactivities another related descriptor, namely, the nucleophilicity excess (Deltaomega(g)-/+) for a nucleophile over the electrophilicity in it, has been defined and tested on all-metal aromatic compounds.

Theoretical study on the complete series of chloroanilines.

The environmental effects of chloroanilines depend on their physical and chemical properties, and it is therefore important to know their structure-property relationships that allow a complete understanding of their environmental consequences. The chemical reactivity profiles of all 19 chloroanilines have been investigated using the density functional theory for the first time. Global reactivity descriptors, such as hardness, chemical potential, electrophilicity index, and polarizability, and local reactivity descriptors, namely, local philicities, have been calculated in order to gain insights into the reactive nature and the reactive sites of the selected systems. Using AIM theory, the presence of hydrogen bond critical points (HBCPs) and the values of electron density and Laplacian of electron density at the HBCPs have been analyzed to appreciate the presence of intramolecular hydrogen bonding in the selected systems. Structure-toxicity analysis of the selected set of chloroanilines demonstrates the importance of the electrophilicity index in the prediction of reactivity/toxicity.

QSPR models for polychlorinated biphenyls: n-Octanol/water partition coefficient.

The logarithmic n-octanol/water partition coefficient (logK(ow)) is an important property for pharmacology, toxicology and medicinal chemistry. Quantitative structure-property relationship (QSPR) model for the lipophilic behaviour (logK(ow)) of the data set containing 133 polychlorinated biphenyl (PCB) congeners is analyzed using the conceptual density functional theory based global reactivity parameter such as electrophilicity index (omega) along with energy of lowest unoccupied molecular orbital (E(LUMO)) and number of chlorine substituents (N(Cl)) as descriptors. A reasonably good coefficient of determination (r(2) = 0.914) and the internal predictive ability (r(cv)(2) = 0.909) values are obtained indicating the significance of the considered descriptors in the property analysis of PCBs. Further, the developed method has widespread applicability from chemical reactivity to toxicity analysis and in studies related to various physicochemical properties in the series of dioxins and other polyaromatic hydrocarbons.

Chemical reactivity indices for the complete series of chlorinated benzenes: solvent effect.

We present a comprehensive analysis to probe the effect of solvation on the reactivity of the complete series of chlorobenzenes through the conceptual density functional theory (DFT)-based global and local descriptors. We propose a multiphilic descriptor in this study to explore the nature of attack at a particular site in a molecule. It is defined as the difference between nucleophilic and electrophilic condensed philicity functions. This descriptor is capable of explaining both the nucleophilicity and electrophilicity of the given atomic sites in the molecule simultaneously. The predictive ability of this descriptor is tested on the complete series of chlorobenzenes in gas and solvent media. A structure-toxicity analysis of these entire sets of chlorobenzenes toward aquatic organisms demonstrates the importance of the electrophilicity index in the prediction of the reactivity/toxicity.

Group philicity and electrophilicity as possible descriptors for modeling ecotoxicity applied to chlorophenols.

The search for the best quantitative structure-activity relationship (QSAR) models in ecotoxicology is an ever-topical research activity. Hence, the development of new descriptors and applying them successfully in QSAR studies seems demanding in ecotoxicology. In the present work, group philicities are utilized for the first time in QSAR analysis for ecotoxicological studies on chlorophenols (CPs). It is important to point out that group philicities are capable of providing the best QSAR model for the toxicity of CPs against Daphnia magna. Furthermore, global electrophilicity and group philicities together give the best QSAR models for Brachydanio rerio and Bacillus with the maximum value of coefficient of determination and high internal predictive ability. The developed QSAR models clearly show the importance of the selected density functional reactivity indices as descriptors in ecotoxicological studies.

Careful scrutiny of the philicity concept.

The philicity concept [J. Phys. Chem. A 2003, 107, 4973] is put in proper perspective. In the present work we analyze different physicochemical problems using philicity. It provides satisfactory results in all such cases. We also compare the relative electro(nucleo)philicity with philicity to show that philicity is better than relative electro(nucleo)philicity when the intermolecular reactivity trends are considered and there is hardly any preference of one above the other as far as the intramolecular reactivities are concerned. On the contrary, the philicity concept has some advantages over the other concept.

pKa prediction using group philicity.

Acid-base dissociation constants (pK(a) values) are important in understanding the chemical, environmental and toxicological properties of molecules. Though various methods have been developed to predict pK(a) by experimental and theoretical models, prediction of pK(a) is still complicated. Hence, a new approach for predicting pK(a) using the group philicity concept has been attempted. Presence of known functional groups in a molecule is utilized as the most important indicator to predict pK(a). The power of this descriptor in describing pK(a) for the series of carboxylic acids, various substituted phenols, anilines, phosphoric acids, and alcohols is probed. Results reveal that the group electrophilicity is suitable for effectively predicting the pK(a) values.