Treatment and follow-up of venous thrombosis in the neonatal intensive care unit: a retrospective study.

OBJECTIVE: The critically ill, premature patients of neonatal intensive care units are susceptible to venous thrombosis, an adverse event associated with short- and long-term morbidity. Venous thrombosis is frequently treated with low-molecular-weight heparins (LMWHs) such as enoxaparin, but optimal dosing of LMWH must balance the morbidity of venous thrombosis with the potential adverse affects of anticoagulation. The optimal dosing of enoxaparin for premature infants is unclear. The objective of this study was to describe enoxaparin therapy and follow-up in critically ill neonates diagnosed with venous thrombosis. STUDY DESIGN: Retrospective medical record review in the neonatal intensive care unit (NICU) in a single tertiary care institution. Infants with venous thrombosis diagnosed in the NICU were identified using preexisting quality improvement lists and medical records. RESULTS: Twenty-six infants with 30 venous thromboses were identified with a median gestational age of 31 weeks at birth. Eighteen (69%) infants received enoxaparin for venous thrombosis during their hospitalization, beginning with a median dose of 1.5 mg kg-1 every 12 h. This dose was increased to a median of 2.1 mg kg-1 every 12 h to achieve target anti-factor Xa levels. The target dose was significantly higher in patients with a postmenstrual age of <37 weeks. Enoxaparin treatment was documented after discharge in 12 patients, continuing for a median of 99 days. Four patients died during hospitalization and their deaths were not attributable to venous thrombosis or anticoagulation complication. Follow-up documentation between 6 and 24 months after venous thrombosis diagnosis revealed no major morbidity of venous thrombosis or enoxaparin therapy. CONCLUSION: Our data reinforce the relative safety and necessity of enoxaparin doses above 1.5 mg kg-1 per 12 h in most neonates. This was particularly true for infants at lower postmenstrual age.

Centrosome-declustering drugs mediate a two-pronged attack on interphase and mitosis in supercentrosomal cancer cells.

Classical anti-mitotic drugs have failed to translate their preclinical efficacy into clinical response in human trials. Their clinical failure has challenged the notion that tumor cells divide frequently at rates comparable to those of cancer cells in vitro and in xenograft models. Given the preponderance of interphase cells in clinical tumors, we asked whether targeting amplified centrosomes, which cancer cells carefully preserve in a tightly clustered conformation throughout interphase, presents a superior chemotherapeutic strategy that sabotages interphase-specific cellular activities, such as migration. Herein we have utilized supercentrosomal N1E-115 murine neuroblastoma cells as a test-bed to study interphase centrosome declustering induced by putative declustering agents, such as Reduced-9-bromonoscapine (RedBr-Nos), Griseofulvin and PJ-34. We found tight 'supercentrosomal' clusters in the interphase and mitosis of ~80% of patients' tumor cells with excess centrosomes. RedBr-Nos was the strongest declustering agent with a declustering index of 0.36 and completely dispersed interphase centrosome clusters in N1E-115 cells. Interphase centrosome declustering caused inhibition of neurite formation, impairment of cell polarization and Golgi organization, disrupted cellular protrusions and focal adhesion contacts-factors that are crucial prerequisites for directional migration. Thus our data illustrate an interphase-specific potential anti-migratory role of centrosome-declustering agents in addition to their previously acknowledged ability to induce spindle multipolarity and mitotic catastrophe. Centrosome-declustering agents counter centrosome clustering to inhibit directional cell migration in interphase cells and set up multipolar mitotic catastrophe, suggesting that disbanding the nuclear-centrosome-Golgi axis is a potential anti-metastasis strategy.

Quantitative multi-parametric evaluation of centrosome declustering drugs: centrosome amplification, mitotic phenotype, cell cycle and death.

Unlike normal cells, cancer cells contain amplified centrosomes and rely on centrosome clustering mechanisms to form a pseudobipolar spindle that circumvents potentially fatal spindle multipolarity (MP). Centrosome clustering also promotes low-grade chromosome missegregation, which can drive malignant transformation and tumor progression. Putative 'centrosome declustering drugs' represent a cancer cell-specific class of chemotherapeutics that produces a common phenotype of centrosome declustering and spindle MP. However, differences between individual agents in terms of efficacy and phenotypic nuances remain unexplored. Herein, we have developed a conceptual framework for the quantitative evaluation of centrosome declustering drugs by investigating their impact on centrosomes, clustering, spindle polarity, cell cycle arrest, and death in various cancer cell lines at multiple drug concentrations over time. Surprisingly, all centrosome declustering drugs evaluated in our study were also centrosome-amplifying drugs to varying extents. Notably, all declustering drugs induced spindle MP, and the peak extent of MP positively correlated with the induction of hypodiploid DNA-containing cells. Our data suggest acentriolar spindle pole amplification as a hitherto undescribed activity of some declustering drugs, resulting in spindle MP in cells that may not have amplified centrosomes. In general, declustering drugs were more toxic to cancer cell lines than non-transformed ones, with some exceptions. Through a comprehensive description and quantitative analysis of numerous phenotypes induced by declustering drugs, we propose a novel framework for the assessment of putative centrosome declustering drugs and describe cellular characteristics that may enhance susceptibility to them.

Induction of robust de novo centrosome amplification, high-grade spindle multipolarity and metaphase catastrophe: a novel chemotherapeutic approach.

Centrosome amplification (CA) and resultant chromosomal instability have long been associated with tumorigenesis. However, exacerbation of CA and relentless centrosome declustering engender robust spindle multipolarity (SM) during mitosis and may induce cell death. Recently, we demonstrated that a noscapinoid member, reduced bromonoscapine, (S)-3-(R)-9-bromo-5-(4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo-[4,5-g]isoquinoline (Red-Br-nos), induces reactive oxygen species (ROS)-mediated autophagy and caspase-independent death in prostate cancer PC-3 cells. Herein, we show that Red-Br-nos induces ROS-dependent DNA damage that resulted in high-grade CA and SM in PC-3 cells. Unlike doxorubicin, which causes double-stranded DNA breaks and chronic G2 arrest accompanied by 'templated' CA, Red-Br-nos-mediated DNA damage elicits de novo CA during a transient S/G2 stall, followed by checkpoint abrogation and mitotic entry to form aberrant mitotic figures with supernumerary spindle poles. Attenuation of multipolar phenotype in the presence of tiron, a ROS inhibitor, indicated that ROS-mediated DNA damage was partly responsible for driving CA and SM. Although a few cells (∼5%) yielded to aberrant cytokinesis following an 'anaphase catastrophe', most mitotically arrested cells (∼70%) succumbed to 'metaphase catastrophe,' which was caspase-independent. This report is the first documentation of rapid de novo centrosome formation in the presence of parent centrosome by a noscapinoid family member, which triggers death-inducing SM via a unique mechanism that distinguishes it from other ROS-inducers, conventional DNA-damaging agents, as well as other microtubule-binding drugs.

Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy.

Nearly a century ago, cell biologists postulated that the chromosomal aberrations blighting cancer cells might be caused by a mysterious organelle-the centrosome-that had only just been discovered. For years, however, this enigmatic structure was neglected in oncologic investigations and has only recently reemerged as a key suspect in tumorigenesis. A majority of cancer cells, unlike healthy cells, possess an amplified centrosome complement, which they manage to coalesce neatly at two spindle poles during mitosis. This clustering mechanism permits the cell to form a pseudo-bipolar mitotic spindle for segregation of sister chromatids. On rare occasions this mechanism fails, resulting in declustered centrosomes and the assembly of a multipolar spindle. Spindle multipolarity consigns the cell to an almost certain fate of mitotic arrest or death. The catastrophic nature of multipolarity has attracted efforts to develop drugs that can induce declustering in cancer cells. Such chemotherapeutics would theoretically spare healthy cells, whose normal centrosome complement should preclude multipolar spindle formation. In search of the 'Holy Grail' of nontoxic, cancer cell-selective, and superiorly efficacious chemotherapy, research is underway to elucidate the underpinnings of centrosome clustering mechanisms. Here, we detail the progress made towards that end, highlighting seminal work and suggesting directions for future research, aimed at demystifying this riddling cellular tactic and exploiting it for chemotherapeutic purposes. We also propose a model to highlight the integral role of microtubule dynamicity and the delicate balance of forces on which cancer cells rely for effective centrosome clustering. Finally, we provide insights regarding how perturbation of this balance may pave an inroad for inducing lethal centrosome dispersal and death selectively in cancer cells.

Differences between adult and pediatric septic shock.

Sepsis is a significant public health problem that affects children and adults alike. Despite some similarities in the approach to pediatric and adult septic shock, there are key differences as it relates to pathophysiology, clinical presentation, and therapeutic approaches. In this review article, we discuss these differences under 4 headings: a) Developmental differences in the hemodynamic response, b) Activated Protein C, c) Thrombocytopenia associated multiple organ failure and d) Hemophagocytic Lymphohistiocytosis (HLH).

A novel microtubule-modulating noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering.

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing 'real' centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the 'cytotoxicity-window' of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some 'apoptosis-evasive' cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation.

Nitric oxide associated with iNOS expression inhibits acetylcholinesterase activity and induces memory impairment during acute hypobaric hypoxia.

The mechanisms responsible for cholinergic dysfunction associated learning and memory impairment during hypoxia are not well-understood. However it is known that inflammatory mediators like inducible nitric oxide synthase (iNOS) hamper the functions of cholinergic neurons. In this present experiment we made an effort to study the iNOS expression mediated retrograde and anterograde memory impairment in Balb/c mice following acute hypobaric hypoxia (at an altitude of 23,000ft for 6h) using elevated plus maze and passive avoidance step-through tasks. Our results demonstrated that hypoxia transiently impairs the retrograde memory without affecting the anterograde memory functions, accompanied with a substantial rise in iNOS expression and nitric oxide levels in cerebral cortex on days 2 and 3 post hypoxia. Treatment with aminoguanidine (iNOS inhibitor ), resulted in down-regulation of the iNOS expression, attenuation of the surge of nitric oxide (NO) in cerebral cortex and reversal of retrograde memory impairment due to hypoxia. Moreover the reduced AChE activity and elevated lipid peroxidation in cerebral cortex were evident during post hypoxia re-oxygenation period, which was not observed in the hippocampus. Additionally, NO donor spermine NONOate could inhibit the AChE activity in brain homogenates in a concentration-dependent manner, which further substantiate that nitric oxide produced during post hypoxia re-oxygenation, primarily contributes to the observed inhibition of cortical AChE activity. Based on these experiments we hypothesize that the NO burst as a result of iNOS upregulation during hypoxia interrupts the memory consolidation by altering the cholinergic functions.

Involvement of angiotensin converting enzyme in cerebral hypoperfusion induced anterograde memory impairment and cholinergic dysfunction in rats.

Forebrain cholinergic dysfunction is the hallmark of vascular dementia (VaD) and Alzheimer's dementia (AD) induced by cerebral hypoperfusion during aging. The aim of the present study is to evaluate the role of angiotensin converting enzyme (ACE) in cerebral hypoperfusion-induced dementia and cholinergic dysfunction. Chronic cerebral hypoperfusion (CHP) was induced by permanent bilateral common carotid artery (2VO) occlusion in rats. Chronic cerebral hypoperfusion resulted in anterograde memory impairment revealed from Morris water maze (MWM) and passive avoidance step through tasks (PA), which was significantly attenuated by ACE inhibitor, captopril. Cerebral hypoperfusion down-regulated the relative expression of cholinergic muscarinic receptor (ChM-1r) and choline acetyltransferase (ChAT) as well as up-regulated the angiotensin II type-1 receptor (AT-1) expression in hippocampus of vehicle treated CHP group on the 54th day post-hypoperfusion. The diminished number of presynaptic cholinergic neurons and the pyramidal neurons were evident from ChAT-immunofluorescence and the hematoxylin and eosin (H&E) staining studies respectively in hippocampal Cornu ammonis1 (CA1); region of vehicle-treated hypoperfused animals. Further the lipid peroxidation level was also found to be elevated in the hippocampus of the vehicle-treated group. Our results demonstrated that continuous captopril treatment (50 mg/kg, i.p. twice daily) for 15 days mitigated the hypoperfusion-induced cholinergic hypofunction and neurodegeneration in hippocampus. The present study robustly reveals that the angiotensinergic system plays a pivotal role in progression of neuronal death and memory dysfunctions during cerebral hypoperfusion.

Modulation of multidrug resistance in cancer cells by the E3 ubiquitin ligase seven-in-absentia homologue 1.

Seven-in-absentia homologue 1 (Siah1) is an E3 ubiquitin ligase that regulates the ubiquitination and proteasome-dependent degradation of a number of proteins. Here we report that Siah1 modulates multidrug resistance 1 (MDR1)/P-glycoprotein-mediated drug resistance in the cancer cell lines examined. Siah1, but not its ligase-dead mutant, down-regulates MDR1/P-glycoprotein and sensitizes the multidrug-resistant cells to chemotherapeutic agents. Mechanistically, Siah1 does not promote P-glycoprotein degradation but decreases its expression transcriptionally by promoting c-Jun transcription factor binding to the activator protein 1 (AP1) site in the MDR1 promoter. Moreover, Siah1 triggers c-Jun NH2-terminal kinase (JNK) activation, leading to enhanced phosphorylation of c-Jun, and the JNK/c-Jun signalling axis is critical for Siah1 to down-regulate MDR1/P-glycoprotein expression. These findings demonstrate a previously unidentified role for Siah1 in regulating MDR1/P-glycoprotein expression through the JNK/c-Jun pathway.

Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site.

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P(2) binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.

Continuous remifentanil for pediatric neurosurgery patients.

OBJECTIVE: Remifentanil hydrochloride is a new titratable opioid agonist with rapid onset and offset of action. We present 2 cases of intracranial mass lesions in whom remifentanil was used to obtain serial neurological examinations. METHODS: Patients received bolus doses of remifentanil 1 microgram/kg followed by continuous infusions of 0.1-0.5 microgram/kg/min, titrated to effect. After the clinical examination, the remifentanil was restarted to the desired effect. CONCLUSION: The ultra-short duration of action of remifentanil makes it ideally suited for selected pediatric neurosurgical patients in whom frequent, serial neurological examinations are necessary. There were no side effects associated with the use of remifentanil.

Antagonistic effect of FePP on the ethanol mediated induction of hepatic, renal and splenic δ-amino levulinic acid synthase activityin vivo in rats.

In the current study, we report perturbations in hepatic, renal and splenic heme synthesis at the level of the rate limiting enzyme, δ-amino levulinic acid synthase (ALA-S) on ethanol administration independently and in conjunction with iron-protoporphyrin (FePP) in male Wistar rats. Excessive ethanol administration (5 ml/kg bw) resulted in a significant induction of hepatic, splenic and renal ALA-S activity. Simultaneous administration of FePP (50 µmol/kg bw) reverted the observed induction response to a sharp decline. The features of the action of ethanol and FePP togetherin vivo, i.e. a substantial inhibition of ALA-S is suggestive of the beneficial effects of this formulation in acute attacks of porphyria.

Inhibitory effect of metalloporphyrins in conjunction with cholesterol on hepatic phospholipase A(2) activityin vivo in rats.

We investigated the effect of cholesterol and the metalloporphyrins cobalt mesoporphyrin (CoMP) and chromium protoporphyrin (CrPP) on phospholipase A(2) (PLA(2)) activity and the consequent hepatic mitochondrial stability as well as on lipid concentrations. Our studies revealed that on administration of cholesterol, CrPP, CoMP as well as simultaneous adminstration of cholesterol and CrPP, there was an inhibition of PLA(2) activity. These moieties may therefore, be agents for preventing destabilisation of the mitochondrial membrane and the consequent pathological conditions which may arise due to membrane lysis. Our results revealed that cholesterol administration increased phospholipid concentration, albeit by modest amounts. Although the independent administration of metalloporphyrins led only to minor elevations in phospholipid concentration, the simultaneous administration of cholesterol and CrPP generated a steep elevation in the concentration of total phospholipid. Since cholesterol inhibits PLA(2) activity it has the potential of being therapeutic agent for preventing the pathological conditions which may arise due to membrane lysis.

An opium alkaloid-papaverine ameliorates ethanol-induced hepatotoxicity: Diminution of oxidative stress.

In this communication, we show the modulatory potential of papaverine, an opium alkaloid and a well known vasodilator agent on the ethanol-induced hepatic oxidative stress in male Wistar rats. Ethanol treatment (50% v/v) enhanced lipid peroxidation significantly accompanied by a decline in the activities of glutathione peroxidase (G-Px), glutathione reductase (GR) and depletion in levels of hepatic glutathione (GSH). Ethanol administration increased hepatic glutathione-s-transferases (GST). Enhanced lipid peroxidation induced by ethanol was significantly reduced when papverine was coadministered (P<0.05). In addition, the depleted levels of glutathione and inhibited activities of G-Px and GR recovered significantly (P<0.05) levelling off to control values on co-exposure. Papaverine (200 mg/kg bw) effectively antagonised the ethanol-induced lipid peroxidation and impaired glutathione levels and glutathione dependent enzyme systems. Our results suggest that papaverine is an effective chemopreventive agent in the liver and may suppress the ethanol-induced hepatotoxicity.

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.

Retinoic acid in association with tin-metalloporphyrins influences heme metabolism in vivo in rats.

In the current study we report the perturbation of key enzymes of the heme metabolic pathway, i.e. delta-amino levulinic acid synthase, heme oxygenase and biliverdin reductase, in vivo by administration of retinoic acid (RA) and retinoic acid in association with tin-metalloporphyrins, viz., tin-protoporphyrin (SnPP) and tin-mesoporphyrin (SnMP) in the liver, spleen, heart and lung of rats. RA at a dosing regimen of 50,000 I.U. stimulated splenic ALA-S activity, whereas co-administration of tin-metalloporphyrins with RA antagonised the RA mediated induction of ALA-S. In the other tissues viz., liver, heart and lung our results showed a diminution of ALA-S activity on RA administration, the level of repression was further attenuated when tin-metalloporphyrins were co-administered with RA. This marked suppression of ALA-S brought forth by concurrent administration of RA and tin-metalloporphyrins is suggestive of the beneficial effect of this formulation in acute attacks of porphyria, similar to heme. Furthermore, our results emphasize that the combined dosing of RA with tin-metalloporphyrins leads to a substantial decline in bilirubin levels due to a profound inhibition of HMOX in the probed tissues. The features of the combined action of RA and tin-metalloporphyrins in vivo lead to a substantial suppression of formation of the potentially toxic metabolite bilirubin, and the enhancement of disposal of the untransformed substrate (heme) of the enzyme that is inhibited. These results define some of the characteristics of a therapeutically useful formulation and represent a new therapeutic approach for the amelioration and management of hyperbilirubinemia.