A low resolution solution structure of the cytokine interleukin-1 beta, a 153 residue protein of molecular weight 17,400, has been determined on the basis of 446 nuclear Overhauser effect (NOE) derived approximate interproton distance restraints involving solely NH, C alpha H and C beta H protons, supplemented by 90 distance restraints for 45 hydrogen bonds, and 79 phi torsion angle restraints. With the exception of 27 C alpha H-C alpha H NOEs, all the NOEs were assigned from a three-dimensional 1H-1H NOE 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum. The torsion angle restraints were obtained from accurate 3JHN alpha coupling constants measured from a HMQC-J spectrum, while the hydrogen bonds were derived from a qualitative analysis of the NOE, coupling constant and amide exchange data. A total of 20 simulated annealing (SA) structures was computed using the hybrid distance geometry-dynamical simulated annealing method. The solution structure of IL-1 beta comprises 12 beta-strands arranged in three pseudo-symmetrical topological units (each consisting of 5 anti-parallel beta-strands), joined by turns, short loops and long loops. The core of the structure, which is made up of the 12 beta-strands, together with the turns joining strands I and II, strands VIII and IX and strands X and XI, is well determined with a backbone atomic root-mean-square (r.m.s.) distribution about the mean co-ordinate positions of 1.2(+/- 0.1) A. The loop conformations, on the other hand, are poorly determined by the current data. A comparison of the core of the low resolution solution structure of IL-1 beta with that of the X-ray structure indicates that they are similar, with a backbone atomic r.m.s. difference of only 1.5 A between the co-ordinates of the restrained minimized mean of the SA structures and the X-ray structure.

The postnatal expression profiles of alpha-sarcomeric actin transcripts and protein are quantified in mouse striated muscles from birth to postnatal day 56 by Northern and Western blot analyses. alpha-Cardiac actin (alpha-CA) transcripts transiently increase between 12 and 21 days after birth in the quadriceps muscle, reaching approximately 90% that found in the adult mouse heart. Although alpha-CA is the alpha-sarcomeric actin isoform expressed in the immature fiber, the expression profiles of other contractile protein isoforms indicate that this postnatal period is not reflective of an immature phenotype. alpha-Skeletal actin (alpha-SA) transcripts accumulate to approximately 32% of the total alpha-sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a simultaneous reappearance of alpha-CA and alpha-SA in postnatal skeletal and heart muscles, respectively, and 2) the contractile protein gene expression profile characteristic of adult skeletal muscle is not achieved until after 42 days postnatal in the mouse. We propose there is a previously uncharacterized period of postnatal striated muscle maturation marked by the reappearance of the minor alpha-sarcomeric actins.

OBJECTIVE

: To determine the effects of therapeutic hypothermia on coagulation parameters during hemorrhagic shock (HS) and fluid resuscitation and on survival, in a rat HS model.

METHODS

: Under light anesthesia and spontaneous breathing, 24 rats underwent HS (phase I) for 90 minutes, during which 2.5 mL/100 g blood was withdrawn over 15 minutes; fluid resuscitation (phase II) for 60 minutes, during which no blood was reinfused but 5.0 mL/100 g lactated Ringer's solution was infused over 30 minutes; and an observation (phase III) without anesthesia until 72 hour. After the volume-controlled hemorrhage, rats were randomized into a hypothermia group (n = 12, 33 degrees C) or a normothermia group (n = 12, 38 degrees C). The rectal temperature in each group was maintained during phases I and II. Whole blood coagulopathy was assessed by Sonoclot analysis (SA) at baseline and the end of phases I and II. Fibrinolysis parameters of thrombin-antithrombin III complex and plasma-alpha-2-plasmin inhibitor complex were also monitored.

RESULTS

: At 72 hour, 10 of 12 hypothermia group rats, and 5 of 12 normothermia group rats remained alive (p < 0.05). Fluid resuscitation significantly decreased hematocrit (20% +/- 5%) compared with baseline (33% +/- 5%; p < 0.05) in all rats. SA showed no significant differences between groups at the end of phase I. However, at the end of phase of II, SA revealed a decreased "clot rate" in hypothermia compared with normothermia (22 clot signal/min +/- 11 clot signal/min vs. 34 clot signal/min +/- 14 clot signal/min; p < 0.05) and a prolonged "time to peak" in hypothermia (15 minutes +/- 5 minutes versus 6 minutes +/- 2 minutes; p < 0.05). No differences in thrombin-antithrombin III complex and plasma-alpha-2-plasmin inhibitor complex values were seen between groups throughout the experiment.

CONCLUSIONS

: Therapeutic mild hypothermia of 33 degrees C did not cause coagulopathy during HS, but did impair SA coagulation parameters during fluid resuscitation, probably because of dilution. Hypothermia also prolonged survival after HS. Impairments to coagulation parameters did not worsen outcomes in the rat HS model.

Social anxiety disorder in adolescents is increasingly recognized as a common condition that may precede onset of other mental health problems. However, few measures are currently available to screen for adolescent social anxiety, and little is known about their psychometric characteristics in school-based samples. To this end, the present study was undertaken as a psychometric cross validation of the Social Anxiety Scale for Adolescents (SAS-A). Exploratory and Confirmatory Factor Analyses (CFA) supported the original 3-factor structure of the SAS-A but retained fewer items than in the original scale. The revised scales demonstrated good internal consistency as well as criterion and concurrent validity. Despite including fewer items, psychometric characteristics of the revised scales were equivalent to or stronger than those reported in previous studies of the measure. As such, the present study provides initial support for the utility of the revised SAS-A as a measure of social anxiety in adolescent school samples.

The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA) to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the "two-pronged plug two-hole socket" model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an alpha-helix. In contrast, a beta-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors.

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR alpha beta + T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4+ or CD8+. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4-CD8- T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4-CD8- T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.

Proinflammatory cytokines have been variably linked to development of cerebral white matter injury (WMI) in preterm infants. Because soluble receptors tightly control cytokine bioactivity, we modeled cytokine-receptor interaction as a predictor of WMI. Plasma from 100 preterm infants was assayed for cytokines (tumor necrosis factor alpha, interleukin (IL-1beta, IL-6) and their soluble receptors (sTNF-RI), sTNF-RII, sIL-1RA, and sIL-6R). Cranial ultrasound (US) results were correlated with cytokine and receptor concentrations individually and with cytokine-receptor interaction models (PROC LOGISTIC; SAS Software). Receiver operating characteristic curves were constructed to determine the predictability of WMI. Fifty-two infants with normal US exams were compared with 21 infants with evidence of WMI. There was no association between individual cytokine or receptor concentrations and the development of WMI. However, modeling cytokines with their soluble receptors significantly improved the predictability of WMI. We concluded that consideration of cytokine-receptor interaction may be more important than individual cytokine concentrations alone in determining the role of inflammation in the pathogenesis of WMI in preterm infants.

OBJECTIVE

Inflammation is known to be a major determinant of the progression of coronary artery disease (CAD). In the present study we have evaluated the plasma levels of cytokines-tumor necrosis factor-alpha (TNF), interleukin- 1 alpha (IL-1), interleukin-6 (IL-6), interferon-gamma (IFN), and interleukin-10 (IL-10)- to examine the association between these cytokines and C-reactive protein (CRP) in patients with CAD.

METHODS

Patients with acute coronary syndromes (ACS; n = 20) were compared with patients with stable angina (SA; n= 20) and with control volunteers (C; n= 20). Blood samples were collected at the time of admission from all patients and 15 and 30 days thereafter.

RESULTS

CRP levels (20.8+/-8.8 mg/L) (mean+/-SEM) were higher at baseline in ACS than SA patients (4.1+/-0.8mg/L) or the control subjects (5.1+/-1.8mg/L) (p<0.05). At admission, IL-6 was detected in 50% of the ACS patients and 5% of the SA patients or control subjects, while TNF was detected in 35% of the ACS and SA patients but only in 5% of control subjects. Subsequently, IL-6 levels declined and were no longer detectable, while TNF levels increased among ACS patients at all time periods tested when compared with other patients. The presence of IL-1 and IL-10 were not detectable in the blood samples examined, and IFN could only be detected in the ACS group. A significant correlation was observed between IL-6 and CRP levels (r=0.4; p<0.01) in all groups. There were no correlations among any of the other cytokines and CRP levels.

CONCLUSIONS

Our study demonstrates raised levels of TNF, IL-6, IFN, and CRP in patients with ACS and a positive correlation between IL-6 and CRP but not with the other cytokines.

Inoue, DS, Panissa, VLG, Monteiro, PA, Gerosa-Neto, J, Rossi, FE, Antunes, BMM, Franchini, E, Cholewa, JM, Gobbo, LA, and Lira, FS. Immunometabolic responses to concurrent training: the effects of exercise order in recreational weightlifters. J Strength Cond Res 30(7): 1960-1967, 2016-The relationship between immunometabolic response and performance is not well understood. This study evaluated the influence of concurrent strength and high-intensity aerobic sequence of exercise order between sessions on strength performance, metabolic, and inflammatory response. Eleven recreational weightlifters underwent the following 2 randomized sessions: (a) strength-aerobic exercise order (SA) and (b) aerobic-strength exercise order (AS). Blood samples were collected before (Pre) and immediately after the first exercise (Post-1) and the second exercise (Post-2) of each session. The SA condition presented a higher number of repetitions (SA: 54 ± 15 vs. AS: 43 ± 12) and total volume (SA: 7,265 ± 2,323 vs. AS: 5,794 ± 1846 kg) than the AS condition (both p = 0.001). Glucose was higher in Pre when compared with post-1 in both orders (p ≤ 0.05); changes in lactate were time-dependent in the different orders (p ≤ 0.05); however, AS post-2 lactate was lower when compared with SA post-2 (p ≤ 0.05). Interleukin-6 levels showed time-dependent changes for both exercise orders (p ≤ 0.05). Tumor necrosis factor alpha (TNF-α) level was increased only in AS post-1 (AS: pre = 21.91 ± 35.47, post-1 = 26.99 ± 47.69 pg·ml vs. SA: pre = 25.74 ± 43.64, post-1 = 29.74 ± 46.05 pg·ml, p ≤ 0.05). These results suggest that concurrent training order exhibits different immunometabolic responses and, at least in part, can be associated with the acute decline in strength performance induced by concurrent exercise. Our results point to a possible role of TNF-α (post-1 AS condition) as a trigger to restore the energy demand by providing substrates to help maintain contractile activity in skeletal muscle.

BACKGROUND

Increasing tumor necrosis factor alpha (TNFalpha) expression is found to be positively associated with spontaneous abortion. TNFalpha acts through the binding of two types of transmembrane receptors called TNFR1 and TNFR2. Here we compare membrane TNFR1 (mTNFR1) protein expression on chorionic villi in women with spontaneous abortion (SA) and viable pregnancy.

METHODS

In a prospective study, 31 women with SA and 30 with a viable pregnancy were studied. Chorionic villous membrane TNFR1 was detected by confocal laser scanning microscopy and immunohistochemistry.

RESULTS

The mTNFR1 protein is expressed in villous stromal, vessel endothelial, syncytiotrophoblast, and cytotrophoblast cells in early pregnancy. The intensity of mTNFR1 fluorescence (mean+/-S.D.) in villous stromal cells was higher in the abortion groups than in the control group (35.99+/-6.35 versus 32.64+/-5.01; p<0.05). In the abortion groups, villous cytotrophoblast cells were intensely positive for mTNFR1, whereas mTNFR1 staining of vessel endothelial and syncytiotrophoblast cells was significantly lower (p<0.001).

CONCLUSIONS

Abundant mTNFR1 expression in the cytotrophoblast cells in women with SA may mediate TNFalpha to induce programmed cell death in the mTNFR1-expressing cytotrophoblasts to limit their growth. The over-expressing mTNFR1 in villous stromal cells, mediating TNFalpha effects, may cause pathological changes or tissue damage in chorionic villi locally in nonviable pregnancies.

K562 cells were used to investigate the factors that control hemoglobin (Hb) synthesis. Treatment with sodium butyrate enhanced Hb synthesis and glycophorin A expression. delta-Aminolevulinate synthase (ALAS) activity and Hb levels simultaneously increased to a similar extent and with a similar time course, and the increases were dependent on the concentration of diferric transferrin (FeTf) in the culture medium. Addition of exogenous delta-aminolevulinic acid (ALA) resulted in a dose-dependent increase in Hb content. Hb synthesis was inhibited 50% after addition of succinylacetone (SA), a potent inhibitor of delta-aminolevulinate dehydratase. These findings suggest that ALAS is a key enzyme in the eight steps of de novo heme synthesis and that iron, including FeTf, plays a central role in Hb synthesis through control of ALAS activity in erythroid differentiating cells. On the other hand, erythropoietin (EPO) treatment had no effect on Hb synthesis and slightly suppressed glycophorin A expression. Hemin enhanced Hb synthesis in the K562 cells but not glycophorin A expression. The addition of ALA, SA, or FeTf to hemin-treated cells caused no significant changes in Hb synthesis. Butyrate, EPO, and hemin acted on the K562 cells in different ways and caused different biochemical changes in the Hb synthesis process.

The objective of this study was to quantify changes of net nutrient metabolism by portal-drained viscera (PDV) and liver of four beef steers (253 +/- 7 kg) in response to combinations of ruminal (R) or abomasal (A) infusions of cornstarch (S) and casein (C). The four treatments in a Latin square design were SACA, SACR, SRCA, and SRCR. Steers were fed alfalfa hay (DMI = 4 kg/d) as a basal diet in 12 equal meals delivered every 2 h and they received continuous infusion of S (800 g/d) and C (200 g/d) in 11-d periods. Digestibilities of DM, N, NDF, and starch, ruminal outflow of liquids and DM, and energy and N retention were less (P < .05) for SA than for SR. Net ammonia and glucose release from PDV were greater (P < .01) for SA than for SR. Net total VFA, acetate, and beta-hydroxybutyrate release from PDV and total splanchnic acetate and beta-hydroxybutyrate release were greater (P < .05) for SR than for SA, but starch infusion site had no effect (P>> .10) on net urea N transfer or alpha-amino N release by PDV. Net release of ammonia N, propionate, and total VFA by PDV and uptake of urea N by PDV were greater for CR than for CA, but net alpha-amino N release by PDV and total splanchnic tissues were greater for CA than for CR (P < .05). Summation of energy supply by PDV indicated no difference in total supply among the treatments, but relative contribution of energy sources was affected by infusion site. Energy release by PDV per unit of DE intake was .68 and .66 for SA and SR, respectively. Net release of glucose by PDV was greater for SACA than for SACR (P < .05). These results suggest that site of digestion of starch and casein varies the relative contribution of nutrients to energy supply by visceral tissues and therefore varies N use in beef steers.

OBJECTIVE

To investigate the structural changes in the cytoskeleton (microtubules, microfilaments) and examine the expression of centrosomal functional proteins during human spermiogenesis.

METHODS

Immunofluorescent staining of human spermiogenic cells.

METHODS

University hospital and IVF clinic.

METHODS

Human testicular tissues were obtained by testicular sperm aspiration (TESA) under informed consent. Three cases of obstructive azoospermia, with confirmed normal spermatogenesis, were examined.

METHODS

Spermatogenic cells were fixed with microtubule-stabilizing buffer. Immunocytochemical detection of microtubules, microfilaments, and centrosome was performed using monoclonal antibodies against alpha- and beta-tubulin, phalloidin, and functional centrosomal proteins.

METHODS

Samples were examined using epifluorescence and laser scanning confocal microscopes.

RESULTS

During the Sb2 period, microtubules formed the manchette structure, which extended from the equator of the nucleus through the cytoplasm. Microfilaments were organized in the periacrosamal region during spermiogenesis (Sa to Sd). Although centrin was observed throughout the spermiogenic period, gamma-tubulin was detected only in the Sb2 period.

CONCLUSIONS

Dynamic cytoskeletal movement was observed during human spermiogenesis. Cytoskeletal rearrangements in the Sb2 period appear to play important roles in the morphologic changes that occur during human spermiogenesis. Studies of the cytoskeletal system during spermiogenesis may help identify some causes of male infertility (e.g., teratozoospermia, maturation arrest).

This report concerns the stepwise biosynthesis in vitro of Sialyl Lewis X, (SA-Le(x)), a carcinoembryonic antigen, in human colon carcinoma KM12 cells exhibiting different metastatic behaviors. The significance of SA-Le(x) has become even more apparent since the detection of its terminal epitope NeuAc(alpha 2-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, as the binding ligand of the selectin family member ELAM-1. The activity level of galactosyltransferase GalT-4 which catalyzes the formation of core nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) is very high in all the metastatic lines tested with highly metastatic lines (KM12-SM) exhibiting the highest activity. The same activity pattern for galactosyltransferase is also observed when tested with iLcOse5Cer (GlcNAc beta 1-3nLcOse4Cer), the precursor for polylactosamine glycolipid. Sialyltransferase SAT-3 which catalyzes the formation of LM1 (NeuAc alpha 2-3nLcOse4Cer), the precursor for SA-Le(x), is also present in all the metastatic cell lines although the activity levels are much lower compared to galactosyltransferase. The fucosyltransferase FucT-3, which catalyzes the formation of R'-Gal-Fuc(alpha 1-3)GlcNAc-R linkage, is active with both nonsialylated substrate, nLcOse4Cer, and sialylated substrate, LM1 (NeuAc alpha 2-3nLcOse4Cer) with the formation of either Le(x) (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc-Cer) or SA-Le(x) (NeuAc alpha 2-3nLcOse4Cer). However, the sialylated substrate LM1 is preferred to enzymatic activity since it exhibited lower Km (46 microM) than that of nLcOse4Cer (67 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To investigate respiratory cycle-related electroencephalographic changes (RCREC) in healthy children and in children with sleep disordered breathing (SDB) during scored event-free (SEF) breathing periods of sleep.

METHODS

Interventional case-control repeated measurements design.

METHODS

Paediatric sleep laboratory in a hospital setting.

METHODS

Forty children with SDB and 40 healthy, age- and sex-matched children.

METHODS

Adenotonsillectomy in children with SDB and no intervention in controls.

RESULTS

Overnight polysomnography; electroencephalography (EEG) power variations within SEF respiratory cycles in the overall and frequency band-specific EEG within stage 2 nonrapid eye movement (NREM) sleep, slow wave sleep (SWS), and rapid eye movement (REM) sleep. Within both groups there was a decrease in EEG power during inspiration compared to expiration across all sleep stages. Compared to controls, RCREC in children with SDB in the overall EEG were significantly higher during REM and frequency band specific RCRECs were higher in the theta band of stage 2 and REM sleep, alpha band of SWS and REM sleep, and sigma band of REM sleep. This between-group difference was not significant postadenotonsillectomy.

CONCLUSIONS

The presence of nonrandom respiratory cycle-related electroencephalographic changes (RCREC) in both healthy children and in children with sleep disordered breathing (SDB) during NREM and REM sleep has been demonstrated. The RCREC values were higher in children with SDB, predominantly in REM sleep and this difference reduced after adenotonsillectomy.

BACKGROUND

Immanuel SA, Pamula Y, Kohler M, Martin J, Kennedy D, Saint DA, Baumert M. Respiratory cycle-related electroencephalographic changes during sleep in healthy children and in children with sleep disordered breathing.

Hypertyrosinemia (HT) is a life-threatening condition caused in large part by the buildup of tyrosine metabolites and their derivatives. One such metabolite is succinylacetone (SA), a potent irreversible inhibitor of heme biosynthesis. Heme is a key component of numerous enzymes involved in arterial blood pressure (BP) regulation, including nitric-oxide synthase (NOS) and its downstream mediator soluble guanylyl cyclase (sGC). Because NOS and sGC are important regulators of cardiovascular function, we hypothesized that inhibition of heme supply to these enzymes by SA would result in the induction of a measurable hypertensive response. Male Sprague-Dawley rats were treated with SA (80 mg x kg(-1) x day(-1) i.p.) for 14 days, resulting in a marked increase in urinary SA and delta-aminolevulinic acid (P < 0.001 for both parameters) and decreased heme concentrations in kidney, liver, spleen, and vascular tissues (P < 0.05 for all parameters). After SA treatment, systemic nitrite/nitrate excretion was reduced by 72% (P < 0.001), and renal NOS and sGC activities were decreased by 32 (P < 0.05) and 38% (P < 0.01), respectively. SA administration also compromised the ex vivo sensitivity of aorta to endothelium-dependent and -independent vasodilation. Despite these effects, SA treatment failed to induce any changes in BP, as assessed by radiotelemetry. Moreover, BP profiles in the SA-treated animals were less responsive to altered sodium intake. The present results demonstrate that extended inhibition of heme synthesis with SA affects hemoenzyme function, albeit without consequent effects on BP regulation and sodium excretion.

Saikosaponin-A (SA), a class of native compound with numerous biological activities, may exert protective effect against postmenopausal bone loss. However, it remains unknown whether SA regulates the osteogenic differentiation of bone marrow stromal cells (BMSCs) in the treatment and prevention of osteoporosis. In this study, BMSCs were treated with various concentrations of SA to stimulate osteogenic differentiation over a 14-day period. Additionally, a canonical ovariectomized (OVX) mouse model was used to evaluate the effect of 3-month SA treatment in preventing postmenopausal osteoporosis. In vitro, we found that SA promotes alkaline phosphatase activity/staining and Alizarin red assay, stimulated the expression of osteogenic markers, i.e., runt-related transcription factor 2 (Runx2), osterix, osteopontin, and osteocalcin (OCN) in BMSCs. In vivo, the trabecular number, trabecular thickness, and trabecular bone mineral density of the distal femoral metaphysis were significantly increased in OVX mice treated intraperitoneally with SA for 3 months compared with OVX mice that not treated with SA. Moreover, the expression of Runx2 and OCN in OVX + SA mice was significantly increased than that in OVX mice. Finally, we found that SA activated the WNT/β-catenin pathway and the expression of several downstream genes including T-cell factor-1 and lymphoid enhancer factor-1. Inhibition of WNT/β-catenin pathway by Dickkopf-related protein 1 blocked the positive role of SA on osteogenesis. Therefore, SA promoted the osteogenic differentiation of BMSCs through WNT/β-catenin signaling.

With the aim of improving the chemotherapeutic index of adenine arabinoside 5-monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes where it is digested in lysosomes. In mice, the L-[3H]SA-ara-AMP conjugates, intravenously injected, selectively penetrated the liver, only small quantities were taken up by cells of spleen, bone marrow, intestine, and brain. After administration of the conjugate to mice with Ectromelia virus hepatitis, ara-AMP was selectively concentrated in liver in a pharmacologically active form. If L-SA-ara-AMP conjugates behave in man as in mouse, their administration to patients with chronic hepatitis B should result in a selective concentration of ara-AMP in liver with a more efficient inhibition of virus replication accompanied by lower toxicity for other tissues.

Quantitative data are presented on the fatty acid composition of rat alpha-fetoprotein (AFP) and serum albumin, (SA), and of brain extracts of suckling rats. In AFP and SA preparations, 40% and 13%, respectively, of total fatty acids present are polyenoic acids. Among them, docosahexaenoic acid is quantitatively the most important in AFP, while in SA, arachidonic acid is largely predominant. Both docosahexaenoic and arachidonic acids were the predominant polyenoic acids in brain extracts. The rate of accumulation of these acids in the brain of suckling rats and the rate of AFP secretion during the same period showed a maximum around 10--12 days after birth. These results suggest that AFP and SA play an important role in the transport and the incorporation of polyunsaturated fatty acids in the developing brain.

Liposomes constructed of egg phosphatidylcholine (EPC), cholesterol (Chol) and stearoylamine (SA) were coated with lectin (Concanavalin-A). These lectinized liposomes were found to retain the ligand binding activity of surface coated concanavalin A (Con-A) as demonstrated by bovine submaxillary mucin (BSM) binding assay. Moreover the ligand specificity of Con-A was maintained even after coating the liposome surface because the presence of competing sugar alpha-methyl mannoside, significantly inhibited the interaction of lectinized liposomes and BSM. The significance of divalent cations for these interactions was studied. The Con-A coating was found to be stable in simulated salivary fluids (SSF, pH 7.2) and under various pH conditions. In vitro targeting studies of lectinized liposomes with gram-negative bacilli (Streptococcus mutans) that harbor in the periodontal pocket (biofilm) demonstrated nearly 100% bacterial growth inhibition (% BGI). The antimicrobial effect was maintained for 360 min. The results were compared with metronidazole bearing plain (protein free/uncoated) liposomes and the free drug at the same dose levels. Mechanisms involved are also discussed. These observations suggest that liposomes coated with lectin (Con-A) were able to maintain the sugar affinity and specificity of the associated ligand and could be targeted to the surface 'glyco-calyx' of bacterial bio-film.

The developmental diversification of neural crest-derived sympathoadrenal (SA) progenitor cells into neuroendocrine adrenal chromaffin cells and sympathetic neurons has been thought to be largely understood. Based on two decades of in vitro studies with isolated SA progenitor and chromaffin cells, it was widely assumed that chromaffin cell development crucially depends on glucocorticoid hormones provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor has revealed that the chromaffin cell phenotype develops largely normally in these mice, except for the induction of the adrenaline synthesizing enzyme phenylethylamine N-methyl transferase. In a search for novel candidate genes that might be involved in triggering the sympathetic neuron/chromaffin cell decision, we have studied putative contributions of transforming growth factor (TGF)-alpha, BMP-4, and the transcription factor MASH-1, molecules with distinct expressions in SA progenitor cells, in their migratory pathways and final destinations. TGF-beta2 and -beta3 and BMP-4 are highly expressed in the wall of the dorsal aorta and in the adrenal anlagen during and after immigration of SA progenitors but expressed at much lower levels in sympathetic ganglia. We found that neutralizing antibodies against all three TGF-beta isoforms applied to the chorionic-allantoic membrane (CAM) of quail embryos interfere with proliferation of immigrated adrenal chromaffin cells but do not affect their specific neuroendocrine ultrastructural phenotype. Grafting of noggin-producing cells to the CAM, which scavenges BMPs, interferes with visceral arch and limb development but does not overtly affect the chromaffin phenotype. The transcription factor MASH-1 promotes early differentiation of SA progenitors. Mice deficient for MASH-1 lack sympathetic ganglia, whereas the adrenal medulla previously has been reported to be present. We show here that most adrenal medullary cells in MASH-1(-/-) mice identified by Phox2b immunoreactivity lack the catecholaminergic marker tyrosine hydroxylase. More surprisingly, most cells do not contain chromaffin granules and display a neuroblast-like ultrastructure and show strongly enhanced expression of c-RET comparable to that observed in sympathetic ganglia. Together, our data suggest that TGF-betas and BMP-4 do not seem to be essential for chromaffin cell differentiation. In contrast with previous reports, however, MASH-1 apparently plays a crucial role in chromaffin cell development.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder, defined clinically by degeneration of dopaminergic neurons and the development of neuronal Lewy bodies. Current treatments of PD are inadequate due to a limited understanding of molecular events of the disease, thus calling for intense research efforts towards identification of novel therapeutic targets. We carried out the present studies towards identifying novel genetic modulators of PD-associated effects employing a transgenic Caenorhabditis elegans model expressing human alpha-synuclein. Employing a systematic RNA interference (RNAi)-based screening approach, we studied a set of neuroprotective genes of C. elegans with an aim of identifying genes that exhibit protective function under alpha-synuclein expression conditions. Our results reveal a novel set of alpha-synuclein effector genes that modulate alpha-synuclein aggregation and associated effects. The identified genes include those from various gene families including histone demethylase, lactate dehydrogenase, small ribosomal subunit SA protein, cytoskeletal protein, collapsin response mediator protein, and choline kinase. The functional characterization of these genes reveals involvement of signaling mechanisms such as Daf-16 and acetylcholine signaling. Further elucidation of mechanistic pathways associated with these genes will yield additional insights into mediators of alpha-synuclein-induced cytotoxicity and cell death, thereby helping in the identification of potential therapeutic targets for PD.

Glycolipid glucuronyltransferase activity (GlcAT-1) has been solubilized and characterized from 19-day-old embryonic chicken brain Golgi-rich membranes. The enzyme catalyzes the biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer glycolipid using neolactetraosylceramide (nLcOse4Cer, Gal beta 1-4GlcNAc beta 1-3Gal beta-1-4Glc-Cer) as the substrate. The membrane-bound enzyme shows optimum activity in the presence of neutral detergents such as Triton CF-54, Triton DF-12, and Nonidet P-40. Approximately 60% of the enzyme activity can be solubilized from the Golgi membrane by Nonidet P-40. The solubilized GlcAT-1 activity is inhibited by different salts such as NaCl, NaBr, NaI, and NaOAc, but not by sodium fluoride (up to 0.4 M concentration). Desialyzed alpha 1 acid glycoprotein (SA alpha 1AGP) can be used as a substrate for glucuronyltransferase. Competition studies between glycolipid (nLcOse4Cer) and glycoprotein SA alpha 1AGP) substrates show a mixed type of inhibition. Phospholipids, in particular phosphatidylglycerol, stimulate solubilized GlcAT-1 activity, while D-erythro-sphingosine, a metabolite of glycosphingolipids, is inhibitory (50% inhibition at 0.8 mM D-erythro-sph). These results demonstrate that both phospholipid as well as sphingosine might be involved in modulating glucuronyltransferase activity.

Scavenging of singlet oxygen (1O2) by alpha-tocopherol (alpha-Toc) was investigated in liposomes. 1O2 was generated by photoirradiation in the presence of two photosensitizers, water-soluble methylene blue (MB) and lipid-soluble 12-(1-pyrene)dodecanoic acid (PDA). The rates of oxidation of alpha-Toc differed depending on the photosensitizing dye and the membrane charge: in the MB-system, alpha-Toc was oxidized fast in negatively charged dimyristoylphosphatidylcholine (DMPC) liposomes containing dicetylphosphate (DCP) and slowly in neutrally charged DMPC liposomes and positively charged DMPC liposomes containing stearylamine (SA), but in the PDA-system, the oxidation rate was independent of the membrane charge. The charge-dependent difference in the MB-system would be due to the site of 1O2 generation depending on the charge-dependent distribution of MB, because positively charged MB increased the zeta-potential of DCP-DMPC liposomes by its interaction with DCP at the membrane surface, but changed the zeta-potentials of DMPC and SA-DMPC liposomes less because of its location in the bulk water phase. The oxidation rate of alpha-Toc in liposomes was different from that in EtOH solution: in the MB system, the oxidation rate was faster in EtOH solution than in DMPC or SA-DMPC liposomes but the same as that in DCP-DMPC liposomes. However, in the PDA system, the oxidation rate was slower in EtOH solution than in DMPC liposomes with or without a charge. Membrane fluidity changed the rate of alpha-Toc oxidation in liposomes, the rate being higher in the liquid crystalline phase than the gel phase, as judged by the higher rate in DMPC liposomes than in dipalmitoylphosphatidylcholine (DPPC) liposomes at 30 degrees C. The rate constants of alpha-Toc for scavenging, the chemical reaction and physical quenching of 1O2 were determined in membranes using DCP-DMPC liposomes labeled with 1,3-diphenyl-isobenzofuran (DPBF), which traps 1O2. These constants differed in the two photosensitizing systems, being higher in the MB-system than in the PDA-system, and were lower than those in EtOH solution.