The mitochondria-targeted antioxidant MitoQ comprises a ubiquinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation. This cation drives the membrane potential-dependent accumulation of MitoQ into mitochondria, enabling the ubiquinol antioxidant to prevent mitochondrial oxidative damage far more effectively than untargeted antioxidants. We sought to fine-tune the hydrophobicity of MitoQ so as to control the extent of its membrane binding and penetration into the phospholipid bilayer, and thereby regulate its partitioning between the membrane and aqueous phases within mitochondria and cells. To do this, MitoQ variants with 3, 5, 10 and 15 carbon aliphatic chains were synthesised. These molecules had a wide range of hydrophobicities with octan-1-ol/phosphate buffered saline partition coefficients from 2.8 to 20000. All MitoQ variants were accumulated into mitochondria driven by the membrane potential, but their binding to phospholipid bilayers varied from negligible for MitoQ3 to essentially total for MitoQ15. Despite the span of hydrophobicites, all MitoQ variants were effective antioxidants. Therefore, it is possible to fine-tune the degree of membrane association of MitoQ and other mitochondria targeted compounds, without losing antioxidant efficacy. This indicates how the uptake and distribution of mitochondria-targeted compounds within mitochondria and cells can be controlled, thereby facilitating investigations of mitochondrial oxidative damage.

Periventricular leukomalacia (PVL) is the major substrate of cerebral palsy in survivors of prematurity. Its pathogenesis is complex and likely involves ischemia/reperfusion in the critically ill premature infant, with impaired regulation of cerebral blood flow, as well as inflammatory mechanisms associated with maternal and/or fetal infection. During the peak period of vulnerability for PVL, developing oligodendrocytes (OLs) predominate in the white matter. We hypothesize that free radical injury to the developing OLs underlies, in part, the pathogenesis of PVL and the hypomyelination seen in long-term survivors. In human PVL, free radical injury is supported by evidence of oxidative and nitrative stress with markers to lipid peroxidation and nitrotyrosine, respectively. Evidence in normal human cerebral white matter suggests an underlying vulnerability of the premature infant to free radical injury resulting from a developmental mismatch of antioxidant enzymes (AOE) and subsequent imbalance in oxidant metabolism. In vitro studies using rodent OLs suggest that maturational susceptibility to reactive oxygen species is dependent, not only on levels of individual AOE, but also on specific interactions between these enzymes. Rodent in vitro data further suggest 2 mechanisms of nitric oxide damage: one involving the direct effect of nitric oxide on OL mitochondrial integrity and function, and the other involving an activation of microglia and subsequent release of reactive nitrogen species. The latter mechanism, while important in rodent studies, remains to be determined in the pathogenesis of human PVL. These observations together expand our knowledge of the role that free radical injury plays in the pathogenesis of PVL, and may contribute to the eventual development of therapeutic strategies to alleviate the burden of oxidative and nitrative injury in the premature infant at risk for PVL.

Control of the HPG axis involves a rapid (30 min) inhibition of LH (GnRH) release by E2. The time course of this effect is faster than expected for a purely transcriptional mechanism of E2 action. To elucidate the mechanism of E2 action, intracellular recordings in TTX were performed in guinea pig hypothalamic GnRH neurons. These neurons were directly hyperpolarized by both the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol, 9 mV) and the GABAB agonist, baclofen (18 mV) by opening K+ channels. Schild analysis with naloxone (Ke = 2.4 nM) confirmed that mu-opioid receptors mediated the effect of DAMGO. E2 also directly hyperpolarized GnRH neurons by opening K+ channels. Coupled with previous work showing a rapid effect of E2 to alter mu-opioid potency (1), a model is presented in which E2 rapidly inhibits GnRH neurons through parallel, possibly synergistic pathways.

Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. Disclosure of potential conflicts of interest is found at the end of this article.

Mature seeds of Arabidopsis thaliana and Brassica napus contain a complex mixture of aliphatic monomers derived from the non-extractable lipid polyesters deposited by various seed tissues. Methods of polyester depolymerization of solvent-extracted seeds and analysis of aliphatic monomers were compared. Sodium methoxide-catalyzed depolymerization, followed by GC analysis of the acetylated monomers, was developed for routine quantitative analysis suitable for 0.5g seed samples. In Arabidopsis seeds, the major C16 and C18 monomers identified included omega-hydroxy fatty acids and alpha,omega-dicarboxylic acids derived from palmitate, oleate and linoleate, and 9,10,18-trihydroxyoctadecenoic acid. Among monomers which can collectively be considered likely to be derived from suberin, docosan-1-ol, docosane-1,22-diol, 22-hydroxydocosanoic acid, 24-hydroxytetracosanoic acid, tetracosane-1,24-dioic acid and ferulic acid were the major species. Compared to Arabidopsis, Brassica seeds showed a roughly similar proportion of monomer classes, with the exception that alkan-1ols were 3-fold higher. Also, there were much less C24 aliphatic species and significant amounts of C14-C16 alkan-1ols, including iso- and anteiso-methyl branched compounds. Dissection and analysis of mature Brassica seeds showed that the trihydroxy C18:1 fatty acid was found mainly in the embryo, while ferulate, fatty alcohols and C22 and C24 species were specific to the seed coat plus endosperm.

Previously, we have shown that the Arabidopsis det2 (deetiolated2) mutant is defective in the biosynthesis of brassinosteroids (BR) and that DET2 (a steroid 5alpha-reductase) acts early in the proposed BR biosynthetic pathway. In this paper we present further biochemical characterization of det2. We have undertaken metabolic experiments with 2H-labeled substrates of intermediates involved in the formation of campestanol from campesterol, and quantitative analysis of intermediates in Arabidopsis wild type and det2. The results of these studies indicate the early operating steps of BR biosynthesis as: campesterol ->> 4-en-3beta-ol ->> 4-en-3-one ->> 3-one ->> campestanol in Arabidopsis, with det2 deficient in the conversion of 4-en-3-one to 3-one. We have also detected these intermediates in the formation of campestanol from campesterol and their metabolic conversions using cultured cells of Catharanthus roseus. These studies confirmed the biosynthetic sequence of events from campesterol to campestanol as was found in Arabidopsis. As such, the originally proposed biosynthetic pathway should be modified.

The nucleus accumbens (NAc) represents a critical site for the rewarding and addictive properties of several classes of abused drugs. The medium spiny GABAergic projection neurons (MSNs) in the NAc receive innervation from intrinsic GABAergic interneurons and glutamatergic innervation from extrinsic sources. Both GABA and glutamate release onto MSNs are inhibited by drugs of abuse, suggesting that this action may contribute to their rewarding properties. To investigate the actions of cannabinoids in the NAc, we performed whole cell recordings from MSNs located in the shell region in rat brain slices. The cannabinoid agonist WIN 55,212-2 (1 microM) had no effect on the resting membrane potential, input resistance, or whole cell conductance, suggesting no direct postsynaptic effects. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were inhibited to a much greater extent by [Tyr-D-Ala(2), N-CH(3)-Phe(4), Gly-ol-enkephalin] (DAMGO, approximately 35%) than by WIN 55,212-2 (<20%), and an analysis of miniature EPSCs suggested that the effects of DAMGO were presynaptic, whereas those of WIN 55,212-2 were postsynaptic. However, electrically evoked GABAergic inhibitory postsynaptic currents (evIPSCs), were reduced by WIN 55,212-2 in every neuron tested (EC(50) = 123 nM; 60% maximal inhibition), and the inhibition of IPSCs by WIN 55,212-2 was completely antagonized by the CB1 receptor antagonist SR141716A (1 microM). In contrast evIPSCs were inhibited in approximately 50% of MSNs by the mu/delta opioid agonist D-Ala(2)-methionine(2)-enkephalinamide and were completely unaffected by a selective mu-opioid receptor agonist (DAMGO). WIN 55,212-2 also increased paired-pulse facilitation of the evIPSCs and did not alter the amplitudes of tetrodotoxin-resistant miniature IPSCs, suggesting a presynaptic action. Taken together, these data suggest that cannabinoids and opioids differentially modulate inhibitory and excitatory synaptic transmission in the NAc and that the abuse liability of marijuana may be related to the direct actions of cannabinoids in this structure.

1. A fast ATP-mediated synaptic current was identified in CA3 pyramidal cells in organotypic hippocampal slice cultures. In the presence of inhibitors for ionotropic glutamate and GABA receptors, extracellular stimulation in the pyramidal cell layer evoked fast synaptic currents that reversed near 0 mV, reflecting an increase in a non-selective cationic conductance. This response was mimicked by focal application of ATP. Antagonists of ionotropic P2X receptors reduced both synaptic and ATP-induced currents. 2. Using a pharmacological approach, the source of synaptically released ATP was determined. Synaptic ATP responses were insensitive to presynaptic blockade of GABAergic transmission between interneurons and CA3 pyramidal cells with the mu-opioid receptor agonist D-Ala(2),MePhe(4),Met(O)(5)-ol-enkephalin (FK33-824), but were blocked by adenosine, which inhibits glutamate release from synaptic terminals in the hippocampus. However, selective inhibition of mossy fibre glutamatergic transmission with the metabotropic glutamate receptor group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) did not affect the response. This result points to the associational fibres as the source of the ATP-mediated synaptic response. 3. These results suggest that ATP, coreleased with glutamate, induces a synaptic response in CA3 pyramidal cells that is observed mainly under conditions of synchronous discharge from multiple presynaptic inputs.

Research has shown that adverse childhood experiences (ACEs) increase the risk of poor health-related outcomes in later life. Less is known about the consequences of ACEs in early adulthood or among diverse samples. Therefore, we investigated the impacts of differential exposure to ACEs on an urban, minority sample of young adults. Health, mental health, and substance use outcomes were examined alone and in aggregate. Potential moderating effects of sex were also explored. Data were derived from the Chicago Longitudinal Study, a panel investigation of individuals who were born in 1979 or 1980. Main-effect analyses were conducted with multivariate logistic and OLS regression. Sex differences were explored with stratified analysis, followed by tests of interaction effects with the full sample. Results confirmed that there was a robust association between ACEs and poor outcomes in early adulthood. Greater levels of adversity were associated with poorer self-rated health and life satisfaction, as well as more frequent depressive symptoms, anxiety, tobacco use, alcohol use, and marijuana use. Cumulative adversity also was associated with cumulative effects across domains. For instance, compared to individuals without an ACE, individuals exposed to multiple ACEs were more likely to have three or more poor outcomes (OR range=2.75-10.15) and four or more poor outcomes (OR range=3.93-15.18). No significant differences between males and females were detected. Given that the consequences of ACEs in early adulthood may lead to later morbidity and mortality, increased investment in programs and policies that prevent ACEs and ameliorate their impacts is warranted.

OBJECTIVE

Ordinary least squares (OLS) regression, commonly called linear regression, is often used to assess, or adjust for, the relationship between a continuous independent variable and the mean of a continuous dependent variable, implicitly assuming a linear relationship between them. Linearity may not hold, however, and analyzing the mean of the dependent variable may not capture the full nature of such relationships. Our goal is to demonstrate how combined use of quantile regression and restricted cubic splines (RCS) can reveal the true nature and complexity of relationships between continuous variables.

METHODS

We provide a review of methodologic concepts, followed by two examples using real data sets. In the first example, we analyzed the relationship between cognition and disease duration in multiple sclerosis. In the second example, we analyzed the relationship between length of stay (LOS) and severity of illness in the intensive care unit (ICU).

RESULTS

In both examples, quantile regression showed that the relationship between the variables of interest was heterogeneous. In the second example, RCS uncovered nonlinearity of the relationship between severity of illness and length of stay.

CONCLUSIONS

Together, quantile regression and RCS are a powerful combination for exploring relationships between continuous variables.

Immature oligodendrocytes (OLs) derive from a large pool of late OL progenitors that populate human cerebral white matter throughout the latter half of gestation. We recently reported that a minor population of immature OLs are present in human cerebral white matter for at least 3 months before these cells commit to myelinogenesis around 30 wk postconceptional age. Since this finding supports dissociation between the events that regulate human immature OL maturation and their commitment to myelinogenesis, we characterized here the cellular sequence of events that characterize immature OLs during the transition from a premyelinating to a myelinating state. Commitment of immature OLs to myelinogenesis in human cerebral white matter correlated with the longitudinal extension of specialized processes, designated "pioneer processes," that made multiple types of apparent contacts with axons. This event coincided with the appearance of 3 distinct populations of sheaths that varied in their labeling for myelin basic protein (MBP). Since few axons initially labeled for MBP, this supported the occurrence in vivo of O4-negative, O1-positive premyelin sheaths that precede MBP-positive compacted myelin. These observations identify 3 sequential stages of early myelinogenesis: 1) the initial ensheathment of axons by premyelin sheaths generated by immature OLs; 2) the initial insertion of MBP into transitional sheaths; and 3) the generation of MBP-rich mature myelin.

The present study investigated whether inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide catabolism, produces antinociception in the acetic acid-induced abdominal stretching model of visceral nociception. Genetic deletion or pharmacological inhibition of FAAH reduced acetic acid-induced abdominal stretching. Transgenic mice that express FAAH exclusively in the nervous system displayed the antinociceptive phenotype, indicating the involvement of peripheral fatty acid amides. The cannabinoid receptor 1 (CB(1)) receptor antagonist, rimonabant, but not the cannabinoid receptor 2 (CB(2)) receptor antagonist, SR144528, blocked the antinociceptive phenotype of FAAH(-/-) mice and the analgesic effects of URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) or OL-135 (1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenyl heptane), respective irreversible and reversible FAAH inhibitors, administered to C57BL/6 mice. The opioid receptor antagonist, naltrexone, did not block the analgesic effects of either FAAH inhibitor. URB597, ED(50) [95% confidence interval (CI) = 2.1 (1.5-2.9) mg/kg], and the nonselective cyclooxygenase inhibitor, diclofenac sodium [ED(50) (95% CI) = 9.8 (8.2-11.7) mg/kg], dose-dependently inhibited acetic acid-induced abdominal stretching. Combinations of URB597 and diclofenac yielded synergistic analgesic interactions according to isobolographic analysis. It is important that FAAH(-/-) mice and URB597-treated mice displayed significant reductions in the severity of gastric irritation caused by diclofenac. URB597 lost its gastroprotective effects in CB(1)(-/-) mice, whereas it maintained its efficacy in CB(2)(-/-) mice, indicating a CB(1) mechanism of action. Taken together, the results of the present study suggest that FAAH represents a promising target for the treatment of visceral pain, and a combination of FAAH inhibitors and NSAIDs may have great utility to treat visceral pain, with reduced gastric toxicity.

OBJECTIVE

Periventricular white matter injury (PWMI) is the major cause of cerebral palsy and cognitive impairment in prematurely born infants. PWMI is characterized by reductions in cerebral myelination and cerebrocortical volumes and is associated with secondary ventriculomegaly. In neonatal rodents, these features of PWMI can be induced by rearing in chronic hypoxia or by activation of A1 adenosine receptors. We determined: (1) whether altered maturation or development of one or more oligodendrocyte (OL) lineage stages plays a role in the pathogenesis of the myelination disturbances associated with exposure to chronic hypoxia, and (2) whether blockade of A1 adenosine receptor action with the adenosine antagonist caffeine can prevent hypoxia-induced white matter injury.

METHODS

Ventriculomegaly and reduced cerebral myelination were generated in mice reared in hypoxia (10% oxygen) from postnatal days 3 (P3) through 12.

RESULTS

Hypomyelination was related to abnormal OL lineage progression and a reduction in the OL progenitor pool. Myelination was enhanced and ventriculomegaly reduced in hypoxia-exposed neonatal pups treated with caffeine from P3 to P12.

CONCLUSIONS

These observations support that hypoxia inhibits OL maturation and that caffeine administration during early postnatal development may have utility in the prevention of PWMI.

The ventral telencephalon in the embryonic brain is thought to provide guidance cues for navigation of thalamocortical axons, but the mechanisms involved remain largely elusive. OL-protocadherin (OL-pc), a member of the cadherin superfamily, is highly expressed by striatal neurons in the developing ventral telencephalon. Here we show that OL-pc-deficient (Pcdh10(-/-)) mice have defects in axon pathways through the ventral telencephalon; for example, thalamocortical and corticothalamic projections cannot cross the ventral telencephalon. In the ventral telencephalon, striatal axons fail to grow out, and, concomitantly, the caudal portion of the globus pallidus and the associated 'corridor' thought to be important for thalamocortical fiber navigation do not form. The inability of the striatum to extend axons is also observed in vitro. These results show that OL-pc is essential for both elongation of striatal axons and patterning of the putative guidance cues for thalamocortical projections.

Progesterone (pregn-4-ene-3,20-dione; P) and its metabolite 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) are secreted by ovaries, adrenals, and glial cells. 3alpha,5alpha-THP in the midbrain ventral tegmental area mediates sexual receptivity of rodents through its actions at GABA(A), NMDA, and/or D(1) receptors. The extent to which 3alpha,5alpha-THP may influence anti-anxiety/anti-stress effects, conditioning and/or reward through these substrates and/or by altering hypothalamic pituitary adrenal axis function is discussed. Biosynthesis of 3alpha,5alpha-THP occurs in responses to mating and may underlie some of the rewarding aspects of sexual behavior. Recent findings from our laboratory which demonstrate that progestins can enhance approach to novel stimuli, conditioning, and reinforcement are reviewed. How progestins' effects on these processes may underlie response to drugs of abuse is considered and new findings which demonstrate interactions between progestins and cocaine are presented.

Periventricular leukomalacia is the predominant injury in the preterm infant leading to cerebral palsy. Oxygen exposure may be an additional cause of brain injury in these infants. In this study, we investigated pathways of maturation-dependent oligodendrocyte (OL) death induced by hyperoxia in vitro and in vivo. Developing and mature OLs were subjected to 80% oxygen (0-24 h). Lactate dehydrogenase (LDH) assay was used to assess cell viability. Furthermore, 3-, 6-, and 10-d-old rat pups were subjected to 80% oxygen (24 h), and their brains were processed for myelin basic protein staining. Significant cell death was detected after 6-24 h incubation in 80% oxygen in pre-OLs (O4+,O1-), but not in mature OLs (MBP+). Cell death was executed by a caspase-dependent apoptotic pathway and could be blocked by the pan-caspase inhibitor zVAD-fmk. Overexpression of BCL2 (Homo sapiens B-cell chronic lymphocytic leukemia/lymphoma 2) significantly reduced apoptosis. Accumulation of superoxide and generation of reactive oxygen species (ROS) were detected after 2 h of oxygen exposure. Lipoxygenase inhibitors 2,3,5-trimethyl-6-(12-hydroxy-5-10-dodecadiynyl-1,4-benzoquinone and N-benzyl-N-hydroxy-5-phenylpentamide fully protected the cells from oxidative injury. Overexpression of superoxide dismutase (SOD1) dramatically increased injury to pre-OLs but not to mature OLs. We extended these studies by testing the effects of hyperoxia on neonatal white matter. Postnatal day 3 (P3) and P6 rats, but not P10 pups, showed bilateral reduction in MBP (myelin basic protein) expression with 24 h exposure to 80% oxygen. Hyperoxia causes oxidative stress and triggers maturation-dependent apoptosis in pre-OLs, which involves the generation of ROS and caspase activation, and leads to white matter injury in the neonatal rat brain. These observations may be relevant to white matter injury observed in premature infants.

Green-leaf volatiles are commonly emitted from mechanically and herbivore-damaged plants. Derived from the lipoxygenase pathway, these compounds may serve as attractants to predators and parasitoids of herbivores, prevent the spread of bacteria and fungi, and induce several important plant defense pathways. In Arabidopsis thaliana, the major volatile released upon mechanical wounding of the leaves is the GLV ester, (Z)-3-hexen-1-yl acetate. We have characterized a member of the BAHD acyltransferase gene family At3g03480 which catalyzes the formation of (Z)-3-hexen-1-yl acetate from acetyl-CoA and (Z)-3-hexen-1-ol. The encoded acetyl CoA:(Z)-3-hexen-1-ol acetyltransferase (CHAT) has the ability to accept several medium-chain-length aliphatic and benzyl-derived alcohols, but has highest catalytic efficiency with (Z)-3-hexen-1-ol. The highest expression of CHAT occurs in the leaves and stems. Upon mechanical damage, the (Z)-3-hexen-1-yl acetate emission peaked after 5 min and declined to pre-damage levels after 90 min. However, CHAT gene transcript levels increased much more slowly with the highest levels detected between 3 and 6 h after wounding. An increase in CHAT enzyme activity in vitro followed the transcript increase, with levels peaking between 10 and 12 h after wounding. Plants expressing either an RNAi cassette for the CHAT gene or plants harboring a T-DNA insertion in the CHAT coding region had greatly reduced (Z)-3-hexen-1-yl acetate emission, showing that the CHAT enzyme is responsible for the in planta production of this ester.

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.

OBJECTIVE

To compare the effects of change from an open to a closed intensive care unit (ICU) format on clinical outcomes, resource utilization, teaching, and perceptions regarding quality of care.

METHODS

Prospective cohort study; prospective economic evaluation.

METHODS

Medical ICU at a university-based tertiary care center. For the open ICU, primary admitting physicians direct care of patients with input from critical care specialists via consultation. For the closed ICU, critical care specialists direct patient care.

METHODS

Consecutive samples of 124 patients admitted under an open ICU format and 121 patients admitted after changing to a closed ICU format. Readmissions were excluded.

METHODS

Comparison of hospital mortality with mortality predicted by the Acute Physiology and Chronic Health Evaluation II (APACHE II) system; duration of mechanical ventilation; length of stay; patient charges for radiology, laboratory, and pharmacy departments; vascular catheter use; number of interruptions of formal teaching rounds; and perceptions of patients, families, physicians, and nurses regarding quality of care and ICU function.

RESULTS

Mean +/- SD APACHE II scores were 15.4 +/- 8.3 in the open ICU and 20.6 +/- 8.6 in the closed ICU (P=.001). In the closed ICU, the ratio of actual mortality (31.4 percent) to predicted mortality (40.1 percent) was 0.78. In the open ICU, the ratio of actual mortality (22.6 percent) to predicted mortality (25.2 percent) was 0.90. Mean length of stay for survivors in the open ICU was 3.9 days, and mean length of stay for survivors in the closed ICU was 3.7 days (P=.79). There were no significant differences between periods in patient charges for radiology, laboratory, or pharmacy resources. Nurses were more likely to say that they were very confident in the clinical judgment of the physician primarily responsible for patient care in the closed ICU compared with the open ICU (41 percent vs 7 percent; P<.Ol), and nurses were the group most supportive of changing to a closed ICU format before and after the study.

CONCLUSIONS

Based on comparison of actual to predicted mortality, changing from an open to a closed ICU format improved clinical outcome. Although patients in the closed ICU had greater severity of illness, resource utilization did not increase.

Quorum-sensing (QS) regulates the production of key virulence factors in Pseudomonas aeruginosa and other important pathogenic bacteria. In this report, extracts of leaves and bark of Combretum albiflorum (Tul.) Jongkind (Combretaceae) were found to quench the production of QS-dependent factors in P. aeruginosa PAO1. Chromatographic fractionation of the crude active extract generated several active fractions containing flavonoids, as shown by their typical spectral features. Purification and structural characterization of one of the active compounds led to the identification of the flavan-3-ol catechin [(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol]. The identity of catechin as one of the active molecules was confirmed by comparing the high-pressure liquid chromatography profiles and the mass spectrometry spectra obtained for a catechin standard and for the active C. albiflorum fraction. Moreover, standard catechin had a significant negative effect on pyocyanin and elastase productions and biofilm formation, as well as on the expression of the QS-regulated genes lasB and rhlA and of the key QS regulatory genes lasI, lasR, rhlI, and rhlR. The use of RhlR- and LasR-based biosensors indicated that catechin might interfere with the perception of the QS signal N-butanoyl-l-homoserine lactone by RhlR, thereby leading to a reduction of the production of QS factors. Hence, catechin, along with other flavonoids produced by higher plants, might constitute a first line of defense against pathogenic attacks by affecting QS mechanisms and thereby virulence factor production.

Oligodendrocytes (OLs) are particularly susceptible to the toxicity of the acute lesion environment after spinal cord injury (SCI). They undergo both necrosis and apoptosis acutely, with apoptosis continuing at chronic time points. Loss of OLs causes demyelination and impairs axon function and survival. In parallel, a rapid and protracted OL progenitor cell proliferative response occurs, especially at the lesion borders. Proliferating and migrating OL progenitor cells differentiate into myelinating OLs, which remyelinate demyelinated axons starting at 2 weeks post-injury. The progression of OL lineage cells into mature OLs in the adult after injury recapitulates development to some degree, owing to the plethora of factors within the injury milieu. Although robust, this endogenous oligogenic response is insufficient against OL loss and demyelination. First, in this review we analyze the major spatial-temporal mechanisms of OL loss, replacement, and myelination, with the purpose of highlighting potential areas of intervention after SCI. We then discuss studies on OL protection and replacement. Growth factors have been used both to boost the endogenous progenitor response, and in conjunction with progenitor transplantation to facilitate survival and OL fate. Considerable progress has been made with embryonic stem cell-derived cells and adult neural progenitor cells. For therapies targeting oligogenesis to be successful, endogenous responses and the effects of the acute and chronic lesion environment on OL lineage cells must be understood in detail, and in relation, the optimal therapeutic window for such strategies must also be determined.

Amphotericin B (AmB)-resistant Leishmania donovani promastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3beta-ol and not ergosterol as in the AmB-sensitive strain.

The transcription factor NF-kappaB regulates the expression of numerous genes controlling the immune and stress responses, inflammatory reaction, cell adhesion, and protection against apoptosis. Incontinentia pigmenti (IP) is the first genetic disorder to be ascribed to NF-kappaB dysfunction. IP is an X-linked dominant genodermatosis antenatally lethal in males. A complex rearrangement of the NEMO (NF-kappaB essential modulator) gene accounts for 85% of IP patients, and results in undetectable NEMO protein and absent NF-kappaB activation. On the other hand, hypohidrotic/anhidrotic ectodermal dysplasia (HED/EDA) has been ascribed to at least three genes also involved in NF-kappaB activation: ectodysplasin (EDA1), EDA-receptor (EDAR) and EDAR-associated death domain (EDARADD). During hair follicle morphogenesis, EDAR is activated by ectodysplasin, and uses EDARADD as an adapter to build a signal transducing complex that leads to NF-kappaB activation. Hence, several forms of HED/EDA also result from impaired activation of the NF-kappaB cascade. Finally, hypomorphic NEMO mutations have been found to cause anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), whilst stop codon mutations cause a more severe phenotype associating EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). The immunological and infectious features observed in patients result from impaired NF-kappaB signalling, including cellular response to LPS, IL-1beta, IL-18, TNF-alpha, Tlr2 and CD40 ligand. Consistently, mouse knockout models have shown the essential role of NF-kappaB in the immune, inflammatory and apoptotic responses. Unravelling the molecular bases of other forms of EDA not associated with mutations in NEMO will possibly implicate other components of the NF-kappaB signalling pathway.

Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.