Molecular hydrogen reacts with the hydroxyl radical, a highly cytotoxic species produced in inflamed tissues. It has been suggested therefore to use gaseous hydrogen in a new anti-inflammatory strategy. We tested this idea, with the aid of the equipment and skills of COMEX SA in Marseille, a group who experiments with oxygen-hydrogen breathing mixtures for professional deep-sea diving. The model used was schistosomiasis-associated chronic liver inflammation. Infected animals stayed 2 weeks in an hyperbaric chamber in a normal atmosphere supplemented with 0.7 MPa hydrogen. The treatment had significant protective effects towards liver injury, namely decreased fibrosis, improvement of hemodynamics, increased NOSII activity, increased antioxidant enzyme activity, decreased lipid peroxide levels and decreased circulating TNF-alpha levels. Under the same conditions, helium exerted also some protective effects, indicating that hydroxyl radical scavenging is not the only protective mechanism. These findings indicate that the proposed anti-inflammatory strategy deserves further attention.

To investigate in humans whether interconversion of leucine and its alpha-keto acid, alpha-ketoisocaproate (KIC), is rate limiting for leucine metabolism, normal volunteers were infused with independent 3H and 14C labels of leucine and KIC. Total leucine carbon (leucine + KIC) entry into the plasma space determined by using both plasma specific activities (SA) of the infused isotopes or by using either plasma SA of the leucine moieties reciprocal to those being infused (e.g., [3H]KIC and [14C]leucine during [3H]leucine and [14C]KIC infusion) were nearly identical. When [3H]leucine and [14C]KIC were infused, the ratio of 3H/14C radioactivity of leucine in plasma proteins was similar both to the ratio of plasma [3H]KIC SA/[14C]leucine SA and to the ratio of the rate of [3H]leucine/[14C]KIC infused. Estimates of leucine oxidation using expired 14CO2 and the plasma SA of the 14C moiety reciprocal to infused [14C]leucine or [14C]KIC were similar. We conclude that the plasma SA of the leucine moiety reciprocal to infused labeled leucine or KIC may reflect more accurately whole-body leucine metabolism in postabsorptive humans.

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on preneoplastic (CL-S1), neoplastic (-SA), and highly malignant (+SA) mouse mammary epithelial cell growth and viability in vitro. Over a 5-day culture period, treatment with 0-120 microM alpha- and gamma-tocopherol had no effect on cell proliferation, whereas growth was inhibited 50% (IC50) as compared with controls by treatment with the following: 13, 7, and 6 microM tocotrienol-rich-fraction of palm oil (TRF); 55, 47, and 23 microM delta-tocopherol; 12, 7, and 5 microM alpha-tocotrienol; 8, 5, and 4 microM gamma-tocotrienol; or 7, 4, and 3 microM delta-tocotrienol in CL-S1, -SA and +SA cells, respectively. Acute 24-hr exposure to 0-250 microM alpha- or gamma-tocopherol (CL-S1, -SA, and +SA) or 0-250 microM delta-tocopherol (CL-S1) had no effect on cell viability, whereas cell viability was reduced 50% (LD50) as compared with controls by treatment with 166 or 125 microM delta-tocopherol in -SA and +SA cells, respectively. Additional LD50 doses were determined as the following: 50, 43, and 38 microM TRF; 27, 28, and 23 microM alpha-tocotrienol; 19, 17, and 14 microM gamma-tocotrienol; or 16, 15, or 12 microM delta-tocotrienol in CL-S1, -SA, and +SA cells, respectively. Treatment-induced cell death resulted from activation of apoptosis, as indicated by DNA fragmentation. Results also showed that CL-S1, -SA, and +SA cells preferentially accumulate tocotrienols as compared with tocopherols, and this may partially explain why tocotrienols display greater biopotency than tocopherols. These data also showed that highly malignant +SA cells were the most sensitive, whereas the preneoplastic CL-S1 cells were the least sensitive to the antiproliferative and apoptotic effects of tocotrienols, and suggest that tocotrienols may have potential health benefits in preventing and/or reducing the risk of breast cancer in women.

A large number of matrix substances have been used for various applications in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The majority of matrices applied in ultraviolet-MALDI MS are crystalline, low molecular weight compounds. A problem encountered with many of these matrices is the formation of hot spots, which lead to inhomogeneous samples, thus leading to increased measurement times and hampering the application of MALDI MS for quantitative purposes. Recently, ionic (liquid) matrices (ILM or IM) have been introduced as a potential alternative to the classical crystalline matrices. ILM are equimolar mixtures of conventional MALDI matrix compounds such as 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (CCA) or sinapinic acid (SA) together with organic bases [e.g., pyridine (Py), tributylamine (TBA) or N,N-dimethylethylenediamine (DMED)]. The present article presents a first overview of this new class of matrices. Characteristic properties of ILM, their influence on mass spectrometric parameters such as sensitivity, resolution and adduct formation and their application in the fields of proteome analysis, the measurement of low molecular weight compounds, the use of MALDI MS for quantitative purposes and in MALDI imaging will be presented. Scopes and limitations for the application of ILM are discussed.

Nicotine, an addictive substance, is the major psychoactive component in cigarette smoke. Both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate (NMDA) receptors are essential for the acute stimulative effects of nicotine on the mesocorticolimbic dopamine system, yet little is known about the effects of chronic nicotine treatment on glutamate receptors. Therefore, we used a model of chronic nicotine self-administration (SA), which emulates important aspects of nicotine intake by humans, to determine whether glutamate receptor subunits were affected. After 18 days of saline vs nicotine SA, ionotropic glutamate receptor subunit levels were determined in brain regions within the mesocorticolimbic system by Western blotting. In prefrontal cortex (PFC), the levels of NMDA receptor subunit 2A (NR2A) and NR2B were increased by 67% (p=0.04) and 83% (p=0.027), respectively. In the ventral tegmental area (VTA), glutamate receptor subunit 2/3 (GluR2/3) increased by 34% (p=0.011). Nicotine SA did not affect the expression of these subunits in dorsal striatum and nucleus accumbens. These findings suggest that chronic nicotine SA selectively increased the levels of ionotropic glutamate receptor subunits in a brain region-specific manner.

We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423-607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311-422). Site-specific mutagenesis revealed that the mutations that either disrupt the alpha-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved approximately 20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4's MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.

OBJECTIVE

The aim of this study was to evaluate several in vitro effects of ultrasound that could revert or prevent the hypoxia, hypovascularity, and hypocellularity observed in osteoradionecrosis.

METHODS

Two different ultrasound machines were evaluated, a "traditional" (1 MHz, pulsed 1:4) and a "long wave" (45 kHz, continuous) machine, tested at various intensities. Ultrasound was applied to human gingival fibroblasts, mandibular osteoblasts, and monocytes. The assays performed were cell proliferation (DNA synthesis), collagen and noncollagenous protein (NCP) synthesis, and cytokine production (ELISA) involving interleukin (IL) 1 beta, IL-6, and IL-8, tumor necrosis factor alpha (TNF alpha), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF).

RESULTS

Both ultrasound machines induced increased cell proliferation in fibroblasts and osteoblasts, between 35% and 52%. The collagen and NCP synthesis were also significantly enhanced to levels up to 112%, the best results being with the 45-kHz machine. The ELISA results showed a slight stimulation of IL-1 beta by all cell types; there was no difference in IL-6 and TNF alpha levels. The angiogenesis-related cytokines evaluated were significantly stimulated: IL-8 and bFGF production was enhanced in osteoblasts, and VEGF production was stimulated in all three cell types. Both ultrasound machines produced the same results, with the recommended intensities being 15 and 30 mW/cm2(SA) for the 45-kHz ultrasound, and 0.1 and 0.4 W/cm2(SAPA) for the 1 MHz ultrasound.

CONCLUSIONS

Therapeutic ultrasound induces in vitro cell proliferation, collagen/NCP production, bone formation, and angiogenesis. These findings support its use in prospective clinical trials for the prevention and treatment of osteoradionecrosis.

Four studies were conducted to develop and validate the Sexual Assertiveness Scale (SAS), a measure of sexual assertiveness in women that consists of factors measuring initiation, refusal, and pregnancy-sexually transmitted disease prevention assertiveness. A total of 1,613 women from both university and community populations were studied. Confirmatory factor analyses demonstrated that the 3 factors remained stable across samples of university and community women. A structural model was tested in 2 samples, indicating that sexual experience, anticipated negative partner response, and self-efficacy are consistent predictors of sexual assertiveness. Sexual assertiveness was found to be somewhat related to relationship satisfaction, power, and length. The community sample was retested after 6 months and 1 year to establish test-retest reliability. The SAS provides a reliable instrument for assessing and understanding women's sexual assertiveness.

Aflatoxin B1 (AFB1) was shown to react primarily with one or more lysine residues in serum albumin (SA), accounting for more than half of the total binding to this protein. The radioactivity associated with SA following administration of [U-14C]AFB1 to rats was cleared with a half-life of 2.5 days, which is not significantly different from the half-life of unmodified albumin in the normal rat. The product isolated from a Pronase digest of in vivo-modified SA was identical by chromatographic retention time and u.v. and mass spectroscopy to the synthetic product obtained by the acylase-catalyzed deacetylation of the reaction product of N alpha-acetyl-L-lysine with 8,9-dihydro-8,9-dibromo-AFB1. The latter was characterized by u.v., fluorescence, 500 MHz 1H-n.m.r. and fast atom bombardment mass spectrometry. The spectral data strongly support a structure in which the terminal dihydrofuran ring of AFB1 has been converted to a pyrrolinone ring. It is proposed that the initial adduct is formed by condensation of the dialdehyde tautomer of 8,9-dihydro-8,9-dihydroxy-AFB1, with the epsilon-amino group of lysine, to form a Schiff base, and that the Schiff base undergoes an Amadori rearrangement to an alpha-amino ketone. The pyrrolinone ring is formed by condensation of the amino group with the remaining aldehyde to yield the final product. The purified product was relatively stable but was shown to decompose significantly under the conditions used to isolate it from modified SA.

Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) from pituitary and chorionic gonadotropin (CG) from placenta are a family of closely related glycoproteins. Each hormone is a heterodimer, consisting of an alpha- and a beta-subunit. Within an animal species, the alpha-subunits of all four glyco-protein hormones have an identical amino acid sequence, whereas each beta-subunit is distinct and confers hormone-specific features to the heterodimer. LH and FSH are synthesized within the same cell, the gonadotroph of the anterior pituitary, but are predominantly stored in separate secretory granules. We have characterized the asparagine-linked oligosaccharides on bovine, ovine and human LH, FSH and TSH. The various pituitary hormones were found to contain unique sulfated oligosaccharides with the terminal sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, sialylated oligosaccharides with the terminal sequence SA alpha Gal beta GlcNAc beta Man alpha, or both sulfated and sialylated structures. Despite synthesis of LH and FSH in the same pituitary cell, sulfated oligosaccharides predominate on LH while sialylated oligosaccharides predominate on FSH for all three animal species. We have examined the reactions leading to synthesis of the sulfated oligosaccharides to determine which steps are hormone specific. The sulfotransferase is oligosaccharide specific, requiring only the sequence GalNAc beta 1----4GlcNAc beta 1----2Man alpha. In contrast, the GalNAc-transferase appears to be protein specific, accounting for the preferential addition of GalNAc to LH, TSH, and free (uncombined) alpha-subunits compared with FSH and other pituitary glycoproteins. The predominance of sulfated oligosaccharide structures on LH may account for sorting of LH and FSH into separate secretory granules. Differences in sulfation and sialylation of LH, FSH and TSH may also play a role in the regulation of hormone bioactivity.

The mechanism of senescence-associated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. p-Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A) and MAPK phosphatase 3 (MKP3). Specific activity of PP1/2A and MKP3 activity significantly decreased during cellular senescence, whereas their protein expression levels did not. To investigate possible mechanism of phosphatase inactivation, we measured reactive oxygen species (ROS) generation by fluorescence-activated cell sorting analysis and found it was much higher in mid-old cells than the young cells. Treating the young cells once with 1 mm H2O2 remarkably induced p-Erk1/2 expression; however, it was transient unless repeatedly treated until 72 h. Multiple treatment of the cells with 0.2 mm H2O2 significantly duplicated inactivation of PP1/2A; however, thiol-specific reagents could reverse the PP1/2A activities, suggesting the oxidation of cysteine molecule in PP1/2A by the increased ROS. When the cells were pretreated with 10 mm N-acetyl-l-cysteine for 1 h, Erk1/2 activation was completely blocked. To elucidate which cysteine residue and/or metal ion in PP1/2A was modified by H2O2, electrospray ionization-tandem mass spectrometry analyses were performed with purified PP1C-alpha and found Cys62-SO3H and Cys105-SO3H, implicating the tertiary structure perturbation. H2O2 inhibited purified PP1C-alpha activity by both oxidation of Cys residues and metal ion(s), evidenced by dithiothreitol and ascorbate-restoration assay. In summary, SA-p-Erk1/2 was most likely due to the oxidation of PP1/2A, which resulted from the continuous exposure of the cells to vast amounts of ROS generated during cellular senescence by oxidation of Cys62 and Cys105 in PP1C-alpha and metal ion(s).

The role of chromosomally encoded toxin-antitoxin (TA) loci in bacterial physiology has been under debate, with the toxin proposed as either an inducer of bacteriostasis or a mediator of programmed cell death (PCD). We report here that ectopic expression of MazF(Sa), a toxin of the TA module from Staphylococcus aureus, led to a rapid decrease in CFU counts but most cells remained viable as determined by differential Syto 9 and propidium iodide staining after MazF(Sa) induction. This finding suggested that the toxin MazF(Sa) induced cell stasis rather than cell death. We also showed that MazF(Sa) selectively cleaves cellular mRNAs in vivo, avoiding "important" transcripts such as recA, gyrB, and sarA mRNAs in MazF(Sa)-induced cells, while these three mRNAs can be cleaved in vitro. The results of Northwestern blotting showed that both sarA and recA mRNAs bind strongly to a putative RNA-binding protein. These data suggest that S. aureus likely undergoes stasis by protecting selective mRNA with RNA-binding proteins upon the expression of MazF(Sa) in vivo.

The plant cell wall is a dynamic cellular compartment consisting of a complex matrix of components that can change dramatically in response to environmental stresses. During pathogen attack, for instance, a wide spectrum of proteins that participate in various sequential processes involved in plant defense is secreted into the cell wall. In this study, a mass spectrometry, data-independent acquisition approach known as LC/MS (E) was used to assess temporal changes in the cell wall proteome in response to different levels of an endogenous inducer of plant disease defense responses, salicylic acid (SA). LC/MS (E) was used as a label-free method that enabled simultaneous protein identification and absolute femtomole quantification of each protein secreted into the extracellular matrix. A total of 74 secreted proteins were identified, 63 of which showed increased specific secretion in response to SA. A majority of this induced secretion occurred within 2 h of treatment, indicating that many proteins are involved in the early stages of plant defenses. We also identified a number of apparently nonclassically secreted proteins, suggesting that, as in many nonplant systems, Golgi/ER-independent mechanisms exist for plant protein secretion. These results provide new insight into plant apoplastic defense mechanisms and demonstrate that LC/MS (E) is a powerful tool for obtaining both relative and absolute proteome-scale quantification that can be applied to complex, time- and dose-dependent experimental designs.

Kv4 channel complexes mediate the neuronal somatodendritic A-type K(+) current (I(SA)), which plays pivotal roles in dendritic signal integration. These complexes are composed of pore-forming voltage-gated alpha-subunits (Shal/Kv4) and at least two classes of auxiliary beta-subunits: KChIPs (K(+)-Channel-Interacting-Proteins) and DPLPs (Dipeptidyl-Peptidase-Like-Proteins). Here, we review our investigations of Kv4 gating mechanisms and functional remodeling by specific auxiliary beta-subunits. Namely, we have concluded that: (1) the Kv4 channel complex employs novel alternative mechanisms of closed-state inactivation; (2) the intracellular Zn(2+) site in the T1 domain undergoes a conformational change tightly coupled to voltage-dependent gating and is targeted by nitrosative modulation; and (3) discrete and specific interactions mediate the effects of KChIPs and DPLPs on activation, inactivation and permeation of Kv4 channels. These studies are shedding new light on the molecular bases of I(SA) function and regulation.

The transcription factor Bach1 heterodimerizes with small Maf proteins to repress Maf recognition element (MARE)-dependent gene expression. The repressor activity of Bach1 is inhibited by the direct binding of heme. To investigate the involvement of Bach1 in the heme-dependent regulation of the expression of the beta-globin gene, mouse erythroleukemia (MEL) cells were cultured with succinylacetone (SA), a specific inhibitor of heme biosynthesis, and the level of beta-globin mRNA was examined. A marked decrease of beta-globin mRNA in SA-treated cells was observed, and this decrease was reversed by the addition of hemin. An iron chelator, desferrioxamine, also lowered the level of beta-globin mRNA. The heme-dependent expression of beta-globin is a transcriptional event since the expression of the human beta-globin gene promoter-reporter gene containing the microlocus control region (microLCR) was inhibited when human erythroleukemia K562 cells and MEL cells were cultured with SA. Hemin treatment restored the decrease in promoter activity caused by SA. The control of the microLCR-beta-globin promoter reporter gene by heme was dependent on DNase I-hypersensitive site 2 (HS2), which contains MARE. The MARE binding activity of Bach1 in K562 and MEL cells increased upon SA treatment, and the increase was diminished by the treatment with hemin. Transient expression of Bach1 suppressed the microLCR activity, and this repressor activity was cancelled by treatment with hemin. The expression of a mutated Bach1 lacking heme-binding sites led to a loss in the heme responsiveness of the microLCR. Furthermore, chromatin immunoprecipitation experiments revealed that Bach1 bound to the MARE of HS2 increased by the treatment of MEL cells with SA, and this was cancelled by hemin. We propose that heme positively regulates the beta-globin gene expression by blocking the interaction of Bach1 with the MARE in the LCR.

Human saliva performs a wide variety of biological functions that are critical for the maintenance of the oral health. Various functions include lubrication, buffering, antimicrobial protection, and the maintenance of mucosal integrity. In addition, whole saliva may be analysed for the diagnosis of human systemic diseases, since it can be readily collected and contains identifiable serum constituents. By using proteomic approach, we have established a reference proteome map of human whole saliva allowing for the resolution of greater than 200 protein spots in a single two-dimensional polyacrylamide gel. Fifty-four protein spots, comprised of 26 different proteins, were identifies using N-terminal sequencing, mass spectrometry, and/or computer matching with protein database. Ten proteins, whose levels were significantly different when bleeding had occurred in the oral cavity, were discussed in this study. These 10 proteins include alpha-1-antrypsin, apolipoprotein A-I, cystatin A, SA, SA-III, and SN, enolase I, hemoglobin beta-chain, thioredoxin peroxiredoxin B, as well as a prolactin-inducible protein. The proteomic approach identifies candidates from human whole saliva that may prove to be of diagnostic and therapeutic significance.

BACKGROUND

There is a paucity of data on the national population-level effectiveness of preventing mother-to-child transmission (PMTCT) programmes in high-HIV-prevalence, resource-limited settings. We assessed national PMTCT impact in South Africa (SA), 2010.

METHODS

A facility-based survey was conducted using a stratified multistage, cluster sampling design. A nationally representative sample of 10 178 infants aged 4-8 weeks was recruited from 565 clinics. Data collection included caregiver interviews, record reviews and infant dried blood spots to identify HIV-exposed infants (HEI) and HIV-infected infants. During analysis, self-reported antiretroviral (ARV) use was categorised: 1a: triple ARV treatment; 1b: azidothymidine >10 weeks; 2a: azidothymidine ≤10 weeks; 2b: incomplete ARV prophylaxis; 3a: no antenatal ARV and 3b: missing ARV information. Findings were adjusted for non-response, survey design and weighted for live-birth distributions.

RESULTS

Nationally, 32% of live infants were HEI; early mother-to-child transmission (MTCT) was 3.5% (95% CI 2.9% to 4.1%). In total 29.4% HEI were born to mothers on triple ARV treatment (category 1a) 55.6% on prophylaxis (1b, 2a, 2b), 9.5% received no antenatal ARV (3a) and 5.5% had missing ARV information (3b). Controlling for other factors groups, 1b and 2a had similar MTCT to 1a (Ref; adjusted OR (AOR) for 1b, 0.98, 0.52 to 1.83; and 2a, 1.31, 0.69 to 2.48). MTCT was higher in group 2b (AOR 3.68, 1.69 to 7.97). Within group 3a, early MTCT was highest among breastfeeding mothers 11.50% (4.67% to 18.33%) for exclusive breast feeding, 11.90% (7.45% to 16.35%) for mixed breast feeding, and 3.45% (0.53% to 6.35%) for no breast feeding). Antiretroviral therapy or >10 weeks prophylaxis negated this difference (MTCT 3.94%, 1.98% to 5.90%; 2.07%, 0.55% to 3.60% and 2.11%, 1.28% to 2.95%, respectively).

CONCLUSIONS

SA, a high-HIV-prevalence middle income country achieved <5% MTCT by 4-8 weeks post partum. The long-term impact on PMTCT on HIV-free survival needs urgent assessment.

Osteoblasts respond to repetitive strain by activating stretch-activated, nonselective cation channels (SA-CAT) and increasing matrix protein production. SA-CAT channels are thought to be responsible for mechano-transduction in osteoblasts, although the molecular identity of the SA-CAT channel has previously been unknown. We have demonstrated that both the UMR-106 osteoblast-like cell line and human osteoblasts in primary culture express the alpha-subunit of the epithelial sodium channel (alpha-ENaC). The ENaC gene product is closely related to a class of proteins that confer touch sensitivity to Caenorhabditis elegans and are referred to as degenerins. A cDNA clone was obtained of the entire coding region of rat alpha-ENaC (alpha-rENaC). Sequence analysis indicated that the osteoblast clone's sequence was identical to that originally cloned from rat colon. The alpha-rENaC cDNA was cloned into an expression plasmid and transfected into LM(TK-) cells, a null cell for SA-CAT activity. Stable transfectants expressed mRNA and the expected 74-kDa protein corresponding to alpha-rENaC. Reconstitution of alpha-rENaC resulted in the expression of a 24.2 +/- 1.0 psec SA-CAT channel (P(Na):P(K) = 1.1 +/- 0.1). The channel is calcium permeable (P(Na):P(Ca) = 1.4 +/- 0.1) and highly selective for cations over anions (P(Na):P(Cl)>>) 20). The channel is only active after negative pressure is applied to cell attached patches, cell swelling, or patch excision. These results represent the first heterologous expression of an SA-CAT channel in a mammalian cell system and provide evidence that the ENaC/degenerin family of proteins are capable of mediating both transepithelial sodium transport and are involved in signal transduction by mechano-sensitive cells such as osteoblasts.

The role of IL-4 in human B cell activation, proliferation, and differentiation was examined. rIL-2, but not rIL-4, was able to promote maximum proliferation and generation of Ig-secreting cells in cultures of highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA). Addition of rIL-4 to rIL-2-supported cultures of SA-stimulated peripheral blood, spleen, or lymph node B cells dramatically suppressed both proliferation and differentiation. Results from experiments in which rIL-4 was added to culture at progressively later times indicated a requirement for rIL-4 to be present during the first 2 days of a 5-day incubation to cause inhibition of responsiveness. When a two-stage culture system was utilized, rIL-4 was found to support proliferation or differentiation of B cells initially activated with SA for 2 days only minimally. However, rIL-4 did not inhibit responses of SA preactivated B cells supported by IL-2. The presence of rIL-4 during the initial 48-h activation of B cells with SA and rIL-2 resulted in a profound inhibition of the ability of the activated B cells to respond subsequently to rIL-2 or lymphokine-rich T cell supernatants. A similar 48-h incubation with rIL-4 alone without SA had no effect on subsequent B cell responsiveness. The presence of rIFN-gamma during B cell activation decreased the inhibitory effect of IL-4. Other cytokines including IFN-alpha, IL-1, and commercially available low m.w. B cell growth factor also diminished the inhibitory effect of IL-4. These results indicate that IL-4 inhibits the capacity of human B cells to be activated maximally by SA and rIL-2 and therefore suggest a new immunomodulatory role for this cytokine.

Cat sartorius has two distinct anatomical portions, anterior (SA-a) and medial (SA-m). SA-a acts to extend the knee and also to flex the hip. SA-m acts to flex both the knee and the hip. The objective of this study was to investigate how a "single motoneuron pool" is used to control at least three separate functions mediated by the two anatomical portions of one muscle. Discharge patterns of single motoneurons projecting to the sartorius muscle were recorded using floating microelectrodes implanted in the L5 ventral root of cats. The electromyographic activity generated by the anterior and medial portions of sartorius was recorded with chronically implanted electrodes. The muscle portion innervated by each motoneuron was determined by spike-triggered averaging of the EMGs during walking on a motorized treadmill. During normal locomotion, SA-a exhibited two bursts of EMG activity per step cycle, one during the stance phase and one during the late swing phase. In contrast, every recorded motoneuron projecting to SA-a discharged a single burst of action potentials per step cycle. Some SA-a motoneurons discharged only during the stance phase, whereas other motoneurons discharged only during the late swing phase. In all cases, the instantaneous frequencygram of the motoneuron was well fit by the rectified smoothed EMG envelope generated by SA-a during the appropriate phase of the step cycle. During normal locomotion, SA-m exhibited a single burst of EMG activity per step cycle, during the swing phase. The temporal characteristics of the EMG bursts recorded from SA-m differed from the swing-phase EMG bursts generated by SA-a.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic amorphous silica (SAS), in the form of pyrogenic (fumed), precipitated, gel or colloidal SAS, has been used in a wide variety of industrial and consumer applications including food, cosmetics and pharmaceutical products for many decades. Based on extensive physico-chemical, ecotoxicology, toxicology, safety and epidemiology data, no environmental or health risks have been associated with these materials if produced and used under current hygiene standards and use recommendations. With internal structures in the nanoscale size range, pyrogenic, precipitated and gel SAS are typical examples of nanostructured materials as recently defined by the International Organisation for Standardisation (ISO). The manufacturing process of these SAS materials leads to aggregates of strongly (covalently) bonded or fused primary particles. Weak interaction forces (van der Waals interactions, hydrogen bonding, physical adhesion) between aggregates lead to the formation of micrometre (μm)-sized agglomerates. Typically, isolated nanoparticles do not occur. In contrast, colloidal SAS dispersions may contain isolated primary particles in the nano-size range which can be considered nano-objects. The size of the primary particle resulted in the materials often being considered as "nanosilica" and in the inclusion of SAS in research programmes on nanomaterials. The biological activity of SAS can be related to the particle shape and surface characteristics interfacing with the biological milieu rather than to particle size. SAS adsorbs to cellular surfaces and can affect membrane structures and integrity. Toxicity is linked to mechanisms of interactions with outer and inner cell membranes, signalling responses, and vesicle trafficking pathways. Interaction with membranes may induce the release of endosomal substances, reactive oxygen species, cytokines and chemokines and thus induce inflammatory responses. None of the SAS forms, including colloidal nano-sized particles, were shown to bioaccumulate and all disappear within a short time from living organisms by physiological excretion mechanisms with some indications that the smaller the particle size, the faster the clearance is. Therefore, despite the new nomenclature designating SAS a nanomaterial, none of the recent available data gives any evidence for a novel, hitherto unknown mechanism of toxicity that may raise concerns with regard to human health or environmental risks. Taken together, commercial SAS forms (including colloidal silicon dioxide and surface-treated SAS) are not new nanomaterials with unknown properties, but are well-studied materials that have been in use for decades.

Herbivore- and jasmonate-induced volatile organic compounds (VOCs), which mediate indirect defense, must provide reliable information for predators that frequently learn to associate their release with feeding herbivores. Yet little is known about variation of these cues within populations of native plants, on a scale encountered by predators. We examined variation in herbivore-elicited VOC emissions and patterns of herbivore-induced jasmonate signaling from accessions of Nicotiana attenuata co-occurring in a native population. VOC emissions elicited by herbivore oral secretions (OS) and by methyl jasmonate (MJ) were characterized using gas chromatography-mass spectrometry (GC-MS), high-resolution two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-ToF-MS) and micro-hydrolysis and micro-hydrogenation reactions. Accessions varied in emissions of abundant (trans-alpha-bergamotene, alpha-duprezianene, trans-beta-ocimene, and cis-3-hexenol) and total detectable VOCs, as well as the accumulation of jasmonates, the jasmonate antagonist salicylic acid (SA), abscisic acid (ABA) and jasmonate signaling-related transcripts after OS elicitation. Yet MJ treatment exacerbated differences in VOC emission, suggesting that much variation in VOC emission is caused by processes downstream of jasmonate signaling. Co-occurring N. attenuata plants emit different VOCs following simulated herbivore elicitation as a result in part of differences in jasmonate production and responsiveness, which could reduce the effectiveness of induced indirect defense.

Eastern equine encephalitis virus (EEEV) causes human encephalitis in North America (NA), but in South America (SA) it has rarely been associated with human disease, suggesting that SA strains are less virulent. To evaluate the hypothesis that this virulence difference is due to a greater ability of NA strains to evade innate immunity, we compared replication of NA and SA strains in Vero cells pretreated with interferon (IFN). Human IFN-alpha, -beta, and -gamma generally exhibited less effect on replication of NA than SA strains, supporting this hypothesis. In the murine model, no consistent difference in IFN induction was observed between NA and SA strains. After infection with most EEEV strains, higher viremia levels and shorter survival times were observed in mice deficient in IFN-alpha/beta receptors than in wild-type mice, suggesting that IFN-alpha/beta is important in controlling replication. In contrast, IFN-gamma receptor-deficient mice infected with NA and SA strains had similar viremia levels and mortality rates to those of wild-type mice, suggesting that IFN-gamma does not play a major role in murine protection. Mice pretreated with poly(I-C), a nonspecific IFN inducer, exhibited dose-dependent protection against fatal eastern equine encephalitis, further evidence that IFN is important in controlling disease. Overall, our in vivo results did not support the hypothesis that NA strains are more virulent in humans due to their greater ability to counteract the IFN response. However, further studies using a better model of human disease are needed to confirm the results of differential human IFN sensitivity obtained in our in vitro experiments.

BACKGROUND

Salvia divinorum (Salvia) is an increasingly popular recreational drug amongst adolescents and young adults. Its primary active ingredient, Salvinorin A (SA)-a highly selective agonist at the κ opiate receptor-is believed to be one of the most potent naturally occurring hallucinogens. However, there is little experimental data on the effects of SA in humans.

METHODS

In a 3-day, double-blind, randomized, crossover, counterbalanced study, the behavioral, subjective, cognitive, psychophysiological, and endocrine effects of 0 mg, 8 mg, and 12 mg of inhaled SA were characterized in 10 healthy individuals who had previously used Salvia.

RESULTS

SA produced psychotomimetic effects and perceptual alterations, including dissociative and somaesthetic effects, increased plasma cortisol and prolactin, and reduced resting electroencephalogram spectral power. The SA administration was associated with a rapid increase of its levels in the blood. SA did not produce euphoria, cognitive deficits, or changes in vital signs. The effects were transient and not dose-related. SA administration was very well-tolerated without acute or delayed adverse effects.

CONCLUSIONS

SA produced a wide range of transient effects in healthy subjects. The perceptual altering effects and lack of euphoric effects would explain its intermittent use pattern. Such a profile would also suggest a low addictive potential similar to other hallucinogens and consistent with κ opiate receptor agonism. Further work is warranted to carefully characterize a full spectrum of its effects in humans, to elucidate the underlying mechanisms involved, and to explore the basis for individual variability in its effects.