Currents through single-ion channels were recorded in the cell-attached configuration from locus ceruleus neurons enzymatically dissociated from newborn rats. When the selective mu opioid receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol was in the patch-clamp electrode, unitary inward currents were observed with conductance of approximately 45 pS (measured at zero pipette potential, with 150 mM potassium in the recording electrode). Long silences, lasting many seconds to minutes, separated periods of activity of similar durations. Within such activity periods the distribution of closed times of the channels was best fitted by the sum of two exponential functions (time constants approximately 1 and 30 ms), and the durations of channel openings were fit by a single exponential function; mean open time increased from 2 to 120 ms as agonist concentration increased. Channel activity was not seen when high concentrations of opioids were applied to the neuron outside the patch-clamp recording electrode, indicating intimate coupling between receptor and potassium channel. Unitary currents with similar properties were also seen when pipettes contained alpha 2 adrenoceptor agonists or somatostatin. Taken with previous findings, the results indicate that mu opioid receptors, alpha 2 adrenoceptors, and somatostatin receptors can couple directly to membrane potassium channels through the local intermediary action of a GTP binding protein.

Differences in the mechanisms underlying tolerance and mu-opioid receptor desensitization resulting from exposure to opioid agonists of different efficacy have been suggested previously. The objective of this study was to determine the effects of protein kinase C (PKC) and G protein-coupled receptor kinase (GRK) inhibition on antinociceptive tolerance in vivo to opioid agonists of different efficacy. A rapid (8-h) tolerance-induction model was used where each opioid was repeatedly administered to naive mice. Animals were then challenged with the opioid after injection of a kinase inhibitor to determine its effects on the level of tolerance. Tolerance to meperidine, morphine, or fentanyl was fully reversed by the PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole (Gö6976). However, in vivo tolerance to [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) was not reversed by PKC inhibition. The novel small-molecule GRK inhibitors beta-adrenergic receptor kinase 1 inhibitor and 2-(8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide (Ro 32-0432) did not reverse the tolerance to meperidine, fentanyl, or morphine but did reverse the tolerance to DAMGO. To correlate GRK-dependent DAMGO-induced tolerance with mu-opioid receptor desensitization, we used in vitro whole-cell patch-clamp recording from mouse locus coeruleus neurons and observed that the GRK inhibitors reduced DAMGO-induced desensitization of mu-opioid receptors, whereas the PKC inhibitor had no effect. These results suggest that tolerance induced by low- and moderate-efficacy mu-opioid receptor agonists is dependent on PKC, whereas tolerance induced by the high-efficacy agonist DAMGO is dependent on GRK.

Female rats take longer to acquire a spatial task during behavioral estrus, when GABA-active progesterone and metabolites are elevated. Whether neurosteroids and neuroactive steroids (neuro(active) steroids), which can act at GABA receptor complexes (GBRs), have activational effects on spatial/reference, working, and long-term memory was investigated. In Experiment 1, ovariectomized Long-Evans rats (N = 107) received oil vehicle or one of six neuro(active) steroids, with varying GBR efficacy (greatest to least efficacious: 5 alpha-pregnan-3 alpha-ol-20-one (THP), 5 alpha-pregnan-3 alpha-ol-11,20-dione, 4-pregnen-3,20-dione 17 alpha-hydroxyprogesterone, 5-pregnen-3 beta-ol-20-one sulfate, and 5-androstan-3 beta-ol-17-one sulfate (DHEAS). Following neuro(active) steroid (3.2 or 6.4 mg/kg) or vehicle sc, rats were tested in a Morris water maze, the following week in a Y maze, and then in an open field. Neuro(active) steroid, but not vehicle, animals had decreased distances to the hidden water maze platform. THP (3.2 and 6.4 mg/kg) animals were faster to find this platform than vehicle animals. In the Y maze, 3.2 mg/kg THP increased percentage correct, but 6.4 mg/kg THP increased latencies to the goal box. DHEAS had the opposite effect, with 3.2 mg/kg increasing latencies to the goal box, while 6.4 mg/kg increased percentage correct. In Experiment 2, N = 75 ovariectomized rats were icv implanted with one of the neuro(active)steroids or cholesterol vehicle and then tested for spatial/reference memory, working and long-term memory, and motoricity/anxiolysis as in Experiment 1. DHEAS implants decreased, while THP increased, latencies and distances to the hidden platform in the Morris water maze. In the Y maze, THP increased latencies and decreased percentage correct, but DHEAS increased the likelihood of correct choice. Open field behavior of animals administered the various neuro(active) steroids (sc or icv) was not different. Thus, of the neuro(active) steroids examined, the neurosteroids THP and DHEAS had the most pronounced activational affects on spatial/reference, working, and long-term memory, independent of motoricity.

OBJECTIVE

Rectus extraocular muscles (EOMs) consist of orbital (OL) and global (GL) layers. This study enumerated the fibers in both layers along the length of each EOM.

METHODS

Four human (ages 17 months-93 years) and three monkey (ages 5-7 years) orbits were serially sectioned in the coronal plane and stained with Masson's trichrome. All fibers of the rectus EOMs were counted using light microscopy at midorbit in all specimens and regular intervals throughout the orbits for one human and one monkey.

RESULTS

In the GL, human EOMs in midorbit contained 8000 to 16,400 fibers, and monkey EOMs contained 3600 to 6600 fibers, varying little among the four rectus EOMs. In humans and monkeys, the number of OL fibers in midorbit varied widely according to specific EOM, being most numerous for the medial rectus (human: 7400-14,600; monkey: 3700-7000). The GL existed over the entire extent of each EOM from origin in the orbital apex into continuity with the tendon inserting on the globe. The OL was absent in the most anterior portion of each EOM, because OL fibers inserted on the respective EOM pulley.

CONCLUSIONS

Primate EOMs contain substantial numbers of OL fibers. Numerical similarity of GL fibers is consistent with similar mechanical loading on each of the four rectus EOMs, as required to rotate the globe. Numerical dissimilarity of OL fibers correlates with varying mechanical loading because of varying elasticities of connective tissues onto which these fibers insert.

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex (Zn2L3+) has been studied (L = alkoxide species of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol). Potentiometric pH titration study disclosed a 1 : 1 phenyl phosphate complexation with Zn2L3+ in aqueous solution. The dissociation constant (= [Zn2L3+][PhOPO3(2-)]/[Zn2L3+-PhOPO3(2-)]) is an extremely small value of 2.5 x 10(-8) mol dm(-3) at 25 degrees C with I = 0.10 (NaNO3). The X-ray crystal analysis of the dizinc(II) complex with p-nitrophenyl phosphate showed that the phosphate dianion binds as a bridging ligand to the two zinc(II) ions.

Omenn's syndrome (OS) is a severe immunodeficiency, characterized by clinical and laboratory features reminiscent of a T helper type-2 (Th2) response. CD30, a member of the tumor necrosis factor receptor superfamily, has been found to be preferentially expressed by human T cell clones exhibiting a Th2-line profile and function. We investigated whether there are derangement in CD30 expression in tissues, and/or abnormalities in soluble CD30 (sCD30) levels in the serum, or both, of three children with OS and one child with maternal engraftment and Omenn's-like syndrome (OLS). Large proportions of tissue-infiltrating T lymphocytes from all four patients expressed CD30, whereas in control tissues, including peripheral blood, CD30+ T lymphocytes were extremely few or absent. In addition, levels of sCD30 were abnormally increased in all patients' sera. T cell clones were generated from sorted CD30+ and CD30-peripheral blood T cells of the patient with OLS who showed unusually high numbers of circulating CD30+ T lymphocytes. Most CD4+ T cell clones derived from CD30+ cells showed a Th2-like cytokine profile, whereas the majority of clones generated from CD30-T cells were Th1. These findings support the hypothesis that Th2 cells are involved in the pathogenesis of OS. Moreover, they provide evidence that detection of CD30+ T cells in tissues, increased levels of sCD30 in biological fluids, or both, reflect the presence of immune responses characterized by prevalent activation of T cells producing Th2 cytokines.

Analysis of heptane-soluble compounds from ovipositors of Heliothis zea and Heliothis virescens shows that both species produce relatively large amounts of (Z)-11-hexadecenal, with traces of (Z)-9-hexadecenal, (Z)-7-hexadecenal, and hexadecanal. Heliothis virescens females differ from Heliothis zea in that they also produce trace amounts of tetradecanal, (Z)-9-tetradecenal, and (Z)-11-hexadecen-1-ol. In both species, trace compounds are essential to pheromonal activity and specificity of chemical signals.

The gamma-aminobutyric acid subtype A (GABAA) receptor is widely considered to be an important target for most clinically effective sedative-hypnotic compounds, including general anesthetics, benzodiazepines, barbiturates, and gaboxadol or THIP (4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3-ol). GABAA receptors are highly expressed in anatomical regions that are implicated in sleep processes, notably the thalamus. Furthermore, concentrations of these drugs that modify behavior in vivo also increase GABA-induced inhibitory conductances in vitro. This review will summarize the functional, regional distribution, and pharmacologic properties of various classes of GABAA receptors. Particular emphasis is placed on subpopulations of GABAA receptors that are expressed in extrasynaptic regions of neurons, as these receptors are exquisitely sensitive to several classes of sedative-hypnotic compounds. Evidence to date suggests that extrasynaptic GABAA receptors can be broadly classified into two groups; those containing the delta subunit and the non-delta subunit-containing GABAA receptors. Finally, the probable contribution of extrasynaptic GABAA receptors in the modulation of sleep will be considered.

Opioid signaling in the nucleus accumbens (NAcc) regulates feeding behavior, having particularly strong effects on consumption of highly palatable foods. Since macronutrient content may contribute to palatability, it is uncertain whether opioid regulation of food consumption is based primarily on its macronutrient content or its flavor per se. In order to isolate the effect of flavor, we manipulated opioid signaling in the NAcc in rats and quantified consumption of differently flavored but nutritionally identical pellets. When pellets of either flavor were presented alone, microinjection of d-Ala(2),N,Me-Phe(4),Gly-ol(5)-enkephalin (DAMGO (a mu opioid receptor (MOP) agonist)) into the NAcc increased consumption of pellets of both flavors equally. When both flavors of pellets were presented simultaneously, however, DAMGO in the NAcc selectively increased, while naltrexone (a non-selective opioid antagonist) in the NAcc selectively decreased, consumption of the more preferred flavor. Systemic naltrexone injection had no flavor specific effects, decreasing consumption of both flavors equally. Non-selective inactivation of NAcc neurons by local microinjection of muscimol (a GABA(A) agonist) increased consumption of both the more- and less-preferred flavors equally. These results indicate that opioid signaling directly regulates a subset of NAcc neurons that can selectively enhance consumption of preferred palatable foods based exclusively on flavor cues.

Suberin in extractive-free potato periderm amounts to approximately 25% determined by NaOCH(3) methanolysis. Monomeric composition is characterized by glycerol (20% of monomers), long-chain alpha, omega-diacids, omega-hydroxyacids, alkanoic acids, and alkan-1-ols, with predominance of octadec-9-enodioic acid and 18-hydroxyoctadec-9-enoic acid (39 and 15% of long-chain monomers, respectively). Aromatic hydroxycinnamyl monomers were also present (<1%). Partial depolymerization of potato periderm suberin using a Ca(OH)(2)-catalyzed methanolysis solubilized approximately 10% of suberin aliphatics. GC-MS analysis showed the presence of monomers, dimers, and trimers (87, 12, and 1% of identified compounds, respectively). A total of 26 dimers were identified by EIMS: monoacylglyceryl esters of alpha,omega-diacids, omega-hydroxyacids, and alkanoic acids (with predominance of the 1- and 2-isomers of the monoacylglyceryl ester of the octadec-9-enodioic acid), as well as feruloyl esters of omega-hydroxyacids and alkan-1-ols and a small quantity of a monoferuloylglycerol. Following a discussion of suberin macromolecular structure, it is proposed that in suberized cell walls, the polyaliphatic polymers have a three-dimensional development ensured by glycerol and exist independently from the associated polyaromatics.

In the present study, a novel and exceptionally sensitive method of high-performance liquid chromatography coupled to coulometric detection, together with concentric dialysis probes, was exploited for an examination of the role of autoreceptors and heteroceptors in the modulation of dopamine, noradrenaline and serotonin levels in single samples of the frontal cortex of freely-moving rats. The selective D3/D2 receptor agonist, CGS 15855A [(+/-)-trans-1,3,4,4a,5,10b-hexahydro-4-propyl-2H-[1]benzopyrano[3 ,4-b]-pyridin-9-ol], and antagonist, raclopride, respectively decreased (-50%) and increased (+60%) levels of dopamine without significantly modifying those of serotonin and noradrenaline. The selective alpha2-adrenergic receptor agonist, dexmedetomidine, markedly decreased noradrenaline levels (-100%) and likewise suppressed those of serotonin and dopamine by -55 and -45%, respectively. This effect was mimicked by the preferential alpha2-adrenergic receptor agonist, guanabenz (-100%, -60% and -50%). Furthermore, the alpha2-adrenergic receptor antagonist, RX 821,002 [2(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline], and the preferential alpha2A-adrenergic receptor antagonist, BRL 44408 [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidaz ole], both evoked a pronounced elevation in levels of noradrenaline (+212%, +109%) and dopamine (+73%, +85%). In contrast, the preferential alpha(2B/2C)-adrenergic receptor antagonist, prazosin, did not modify noradrenaline and dopamine levels. RX 821,002 and BRL 44408 did not significantly modify levels of serotonin, whereas prazosin decreased these levels markedly (-55%), likely due to its alpha1-adrenergic receptor antagonist properties. The selective serotonin-1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), reduced serotonin levels (-65%) and increased those of dopamine and noradrenaline by +100%), and +175%, respectively. The selective serotonin-1A antagonist, WAY 100,635 [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo- hexanecarboxamide], which had little affect on monoamine levels alone, abolished the influence of 8-OH-DPAT upon serotonin and dopamine levels and significantly attenuated its influence upon noradrenaline levels. Finally, the selective serotonin-1B agonist, GR 46611 [3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl)acrylamid e], decreased serotonin levels (-49%) and the serotonin-1B antagonist, GR 127,935 [N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-2'-methyl-4'-(5-me thyl-1,2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide], which did not significantly modify serotonin levels alone, abolished this action of GR 46611. Levels of dopamine and noradrenaline were not affected by GR 46611 or GR 127,935. In conclusion, there is a complex pattern of reciprocal autoreceptor and heteroceptor control of monoamine release in the frontal cortex. Most notably, activation of alpha2-adrenergic receptors inhibits the release of noradrenaline, dopamine and serotonin in each case, while stimulation of serotonin-1A receptors suppresses serotonin, yet facilitates noradrenaline and dopamine release. In addition, dopamine D2/D3 autoreceptors restrain dopamine release while (terminal-localized) serotonin-1B receptors reduce serotonin release. Control of serotonin release is expressed phasically and that of noradrenaline and dopamine release tonically.

The injection of neurotensin or the enkephalin analog Tyr-D-Ala-Gly-MePhe-Gly(ol) (DAMGO) into the A10 region of rats produces a motor stimulant effect that is associated with an increase in the postmortem levels of dopamine metabolites in the nucleus accumbens. These behavioral and neurochemical effects are augmented following daily administration. In vivo dialysis in the nucleus accumbens of conscious rats was used to determine if the acute and augmented behavioral responses following neurotensin or DAMGO administration are associated with an increase in extracellular dopamine concentrations. The acute injection of DAMGO produced a dose-dependent (0.03-3.3 nmol) elevation in extracellular dopamine and its metabolites in the nucleus accumbens. Neurotensin also produced a dose-related (0.1-3.3 nmol) increase in dopamine and its metabolites. The elevation in extracellular dopamine produced by DAMGO, but not by neurotensin, was positively correlated with the increase in motor activity. Following daily treatment of either neurotensin (1.0 nmol X 4 d) or DAMGO (0.03 nmol X 4 d), a significant elevation in extracellular dopamine levels occurred in the nucleus accumbens compared to an acute injection. The time course of the change in extracellular dopamine after daily injections was similar to the time course of the behavioral stimulation for both compounds. These data demonstrate that enhanced dopamine release in the nucleus accumbens mediates the acute behavioral effect of DAMGO but does not entirely explain the motor effects of neurotensin. However, enhanced dopamine release may mediate the behavioral sensitization produced by daily injection of both peptides into the A10 region.

The role of flavonoids in CVD, especially in strokes, is unclear. Our aim was to study the role of flavonoids in CVD. We studied the association between the intakes of five subclasses (flavonols, flavones, flavanones, flavan-3-ols and anthocyanidins), a total of twenty-six flavonoids, on the risk of ischaemic stroke and CVD mortality. The study population consisted of 1950 eastern Finnish men aged 42-60 years free of prior CHD or stroke as part of the prospective population-based Kuopio Ischaemic Heart Disease Risk Factor Study. During an average follow-up time of 15.2 years, 102 ischaemic strokes and 153 CVD deaths occurred. In the Cox proportional hazards model adjusted for age and examination years, BMI,systolic blood pressure, hypertension medication, serum HDL- and LDL-cholesterol, serum TAG, maximal oxygen uptake, smoking, family history of CVD, diabetes, alcohol intake, energy-adjusted intake of folate, vitamin E, total fat and saturated fat intake (percentage of energy), men in the highest quartile of flavonol and flavan-3-ol intakes had a relative risk of 0.55 (95% CI 0.31, 0.99) and 0.59 (95% CI 0.30, 1.14) for ischaemic stroke, respectively, as compared with the lowest quartile. After multivariate adjustment, the relative risk for CVD death in the highest quartile of flavanone and flavone intakes were 0.54 (95% CI 0.32, 0.92) and 0.65 (95% CI 0.40, 1.05), respectively. The present results suggest that high intakes of flavonoids may be associated with decreased risk of ischaemic stroke and possibly with reduced CVD mortality.

As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant defense responses against insect pests and pathogens. However, how plants employ their specific defense responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant-specific defense responses. The loss-of-function mutant hpl3-1 produced disease-resembling lesions spreading through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity, hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3-1 plants exhibited enhanced induction of JA, trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including (Z)-3-hexen-1-ol. OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stål)] but negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover, hpl3-1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild-type, most likely as a result of increased release of BPH-induced volatiles. Interestingly, hpl3-1 plants also showed increased resistance to bacterial blight (Xanthomonas oryzae pv. oryzae). Collectively, these results indicate that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice-specific defense responses against different invaders.

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Flavonoids are found in a variety of foods and have anticarcinogenic properties in experimental models. Few epidemiologic studies have examined whether flavonoid intake is associated with breast cancer in humans. In this study, the authors investigated whether dietary flavonoid intake was associated with reduced risk of breast cancer in a population-based sample of US women. They conducted a case-control study among women who resided in Nassau and Suffolk counties on Long Island, New York. Cases and controls were interviewed about known and suspected risk factors and asked to complete a food frequency questionnaire regarding their average intake in the prior 12 months. A total of 1,434 breast cancer cases and 1,440 controls provided adequate responses. A decrease in breast cancer risk was associated with flavonoid intake; the decrease was most pronounced among postmenopausal women for flavonols (odds ratio (OR) = 0.54, 95% confidence interval (CI): 0.40, 0.73), flavones (OR = 0.61, 95% CI: 0.45, 0.83), flavan-3-ols (OR = 0.74, 95% CI: 0.55, 0.99), and lignans (OR = 0.69, 95% CI: 0.51, 0.94). The authors conclude that intake of flavonols, flavones, flavan-3-ols, and lignans is associated with reduced risk of incident postmenopausal breast cancer among Long Island women. These results suggest that US women can consume sufficient levels of flavonoids to benefit from their potential chemopreventive effects.

One of the more surprising recent discoveries in glial biology has been that oligodendrocytes (OLs) originate from very restricted regions of the embryonic neural tube. This was surprising because myelinating OLs are widespread in the mature central nervous system, so there was no reason to suspect that their precursors should be restricted. What we now know about early OL development suggests that they might have as much (or more) in common with ventral neurons-specifically motor neurons (MNs)-as with other types of glia. This has implications for the way we think about glial development, function, and evolution. In this article we review the evidence for a shared MN-OL lineage and debate whether this is the only lineage that generates OLs. We decide in favour of a single embryonic lineage with regional variations along the anterior-posterior neuraxis.

Myelin, formed by oligodendrocytes (OLs) in the CNS, is critical for axonal functions, and its damage leads to debilitating neurological disorders such as multiple sclerosis. Understanding the molecular mechanisms of myelination and the pathogenesis of human myelin disease has been limited partly by the relative lack of identification and functional characterization of the repertoire of human myelin proteins. Here, we present a large-scale analysis of the myelin proteome, using the shotgun approach of 1-dimensional PAGE and liquid chromatography/tandem MS. Three hundred eight proteins were commonly identified from human and mouse myelin fractions. Comparative microarray analysis of human white and gray matter showed that transcripts of several of these were elevated in OL-rich white matter compared with gray matter, providing confidence in their detection in myelin. Comparison with other databases showed that 111 of the identified proteins/transcripts also were expressed in OLs, rather than in astrocytes or neurons. Comparison with 4 previous myelin proteomes further confirmed more than 50% of the identified proteins and revealed the presence of 163 additional proteins. A select group of identified proteins also were verified by immunoblotting. We classified the identified proteins into biological subgroups and discussed their relevance in myelin biogenesis and maintenance. Taken together, the study provides insights into the complexity of this metabolically active membrane and creates a valuable resource for future in-depth study of specific proteins in myelin with relevance to human demyelinating diseases.

The health benefits associated with tea consumption have resulted in the wide inclusion of green tea extracts in botanical dietary supplements, which are widely consumed as adjuvants for complementary and alternative medicines. Tea contains polyphenols such as catechins or flavan-3-ols including epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCG), as well as the alkaloid, caffeine. Polyphenols are antioxidants, and EGCG, due to its high levels, is widely accepted as the major antioxidant in green tea. Therefore, commercial green tea dietary supplements (GTDS) may be chemically standardized to EGCG levels and/or biologically standardized to antioxidant capacity. However, label claims on GTDS may not correlate with actual phytochemical content or antioxidant capacity nor provide information about the presence and levels of caffeine. In the current study, 19 commonly available GTDS were evaluated for catechin and caffeine content (using high-performance liquid chromatography) and for antioxidative activity [using trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays]. Product labels varied in the information provided and were inconsistent with actual phytochemical contents. Only seven of the GTDS studied made label claims of caffeine content, 11 made claims of EGCG content, and five specified total polyphenol content. Caffeine, EGCG, and total polyphenol contents in the GTDS varied from 28 to 183, 12-143, and 14-36% tablet or capsule weight, respectively. TEAC and ORAC values for GTDS ranged from 187 to 15340 and from 166 to 13690 mumol Trolox/g for tablet or capsule, respectively. The antioxidant activities for GTDS determined by TEAC and ORAC were well-correlated with each other and with the total polyphenol content. Reliable labeling information and standardized manufacturing practices, based on both chemical standardization and biological assays, are recommended for the quality control of botanical dietary supplements.

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification (1-3). While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy & de Vellis (2) is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published (4-6), a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

BACKGROUND

Generalized linear models (GLMs) have recently been introduced into cost data analysis. GLMs, transformations of the linear regression model, are characterized by a particular response distribution from one of the exponential family of distributions and monotonic link function which relates the response mean to a scale on which additive model effects operate.

OBJECTIVE

This study compared GLMs and ordinary least squares regression (OLS) in predicting individual patient costs in adult intensive care units (ICUs) and sought to define the utility of the inverse Gaussian distribution family within GLMs.

METHODS

A prospective 'ground-up' utilization costing study was performed in three adult university associated ICUs, enrolling consecutive ICU admissions over a 6-month period in 1991. ICU utilization, patient demographic and ICU admission day data were recorded by dedicated data collectors. Model performance was assessed by prediction error [mean absolute error (MAE), root mean squared error (RMSE)] and residual analysis.

RESULTS

The cohort, 1098 patients surviving ICU, was of mean (SD) age 56 (19.5) years and 41% female. Patient costs per ICU episode (1991 A$) were A$6311 (9689), with range A$106 to A$95602. Prediction error for mean costs was minimal (MAE 4780; RMSE 8965) with OLS using heteroscedastic retransformation of log costs and GLM with Gaussian family and log link (MAE 4798; RMSE 8907). Residual analysis suggested optimal overall performance for the above two models and a GLM with inverse Gaussian family and log link.

CONCLUSIONS

Traditional cost models of OLS with (log) cost transformation may be supplemented by appropriately specified GLM which more closely model the error structure.

The identification of mechanisms associated with phenolic neuroprotection is delayed due to a lack of information regarding the ability of phenolic compounds to enter the central nervous system (CNS). The aim of this work was to evaluate the transmembrane transport of catechin and epicatechin across blood-brain barrier (BBB). Two BBB cell lines, RBE-4 cells (immortalized cell line of rat capillary cerebral endothelial cells) and hCMEC/D3 (immortalized human cerebral microvessel endothelial cell line), were used. HPLC-DAD/MS was used to detect these compounds and their metabolites in the studied samples. The metabolites of the tested flavan-3-ols were synthesized to be used as standards. Catechin and epicatechin could cross both cells in a time-dependent manner. This transport was stereoselective (epicatechin ≫ catechin), involving one or more stereoselective entities. Additionally, these cells were capable of metabolizing these compounds, particularly by conjugation with glucuronic acid, since this metabolite was detected in the basolateral media. Several studies suggest that blood levels of catechin and epicatechin are far below the levels used in this study and that these compounds appeared mainly as methyl, sulfate and glucuronide metabolites. Nevertheless, the information obtained by this study is valuable for the new insights about flavan-3-ols transport.

CONCLUSIONS

(i) catechin and epicatechin are capable of crossing the BBB; (ii) a stereoselective process was involved in the passage of these compounds across BBB cells; (iii) these endothelial cells have enzymes capable of metabolizing these compounds.

Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor (IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in studies of the role of the 16,17-double bond in its mechanism of action, the novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and 17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate 16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of 6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19 (IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent (IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation of the inhibitor with the enzyme. Inhibition following preincubation with these compounds is not lessened by dialysis for 24 h, implying irreversible binding to the enzyme. In contrast under these conditions the still potent (IC50 27 nM) 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h of dialysis and complete reversal of inhibition after 24 h. This behavior was also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24. Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double bond, the inhibition of the enzymic reaction was not enhanced by preincubation either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack this structural feature. The results show that the 16,17-double bond is necessary for irreversible binding of these pyridyl steroids to cytochrome P45017alpha. However oxidation to an epoxide is probably not involved since epoxide 6 was only a moderately potent inhibitor (IC50 260 nM).

Hypoxia-ischemia (HI) is a leading cause of white matter damage, a major contributor to cerebral palsy in premature infants. Preferential white matter damage is believed to result from vulnerability of the immature oligodendrocyte (the pro-OL) to factors elevated during ischemic damage, such as oxygen free radicals and glutamate. In order to determine whether pro-OLs undergo apoptotic death after HI, we analyzed periventricular white matter OLs in P7 rats 4, 12 and 24 h after HI to analyze the time course and mode of cell death. DNA fragmentation was seen at 12 and 24 h of recovery after HI, representing a 17-fold increase over control. In addition, caspase-3 activation was found in NG2+ pro-OLs at 12 h. Electron-microscopic analysis of cell death in the white matter revealed a transition from early necrotic deaths to hybrid cell deaths to classical apoptosis between 4 and 24 h of recovery from HI. The delayed time course of apoptosis in pro-OLs supports the feasibility of interventions to improve clinical outcomes for newborns surviving birth asphyxia.