Neutrophils represent an important source of autoantigens for antineutrophil cytoplasmic antibody associated with vasculitis. To date, two cytoskeletal proteins, vinculin and vimentin, have been reported to be expressed on the cell surfaces of activated macrophages, platelets, and apoptotic T lymphocytes. However, such cell surface expression has never been studied in human neutrophils. As we recently demonstrated that different cytoskeletal proteins were cleaved in apoptotic neutrophils, we hypothesized that some of these were expressed on the cell surface of apoptotic neutrophils. Herein, we found that among vinculin, paxillin, gelsolin, vimentin, lamin B1, alpha-tubulin, and beta-tubulin, only the two intermediate filament (INFIL) proteins, vimentin and lamin B1, are expressed on the cell surface of 24-h aged neutrophils [spontaneous apoptosis (SA)]. By monitoring intracellular expression of vimentin and lamin B1 during SA, we found that these two proteins were cleaved and that such cleavage was reversed by the pan caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk). When neutrophil apoptosis was delayed or suppressed by lipopolysaccharide or the cytokines granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, or interleukin-4, the loss of intracellular expression of vimentin and lamin B1 was prevented. The INFIL proteins were absent from the cell surface when neutrophil apoptosis was delayed. Addition of z-VAD-fmk significantly decreased the cell surface expression of vimentin and lamin B1 during SA. This study provides the first evidence that apoptotic neutrophils express cytoskeletal proteins on their surface, opening the possibility that these cells may participate in the development of autoantibodies directed against cytoskeletal proteins, a condition frequently reported in several inflammatory diseases.

Human and animal influenza A isolates of the H3 serotype preferentially bind SA alpha 2,6Gal or SA alpha 2,3Gal linkages (where SA represents sialic acid), respectively, on cell-surface sialyloligosaccharides. Previously, we have demonstrated selection of SA alpha 2,3Gal-specific receptor variants of several human viruses which differed from the parent viruses by a single amino acid at residue 226 of the hemagglutinin which is located in the receptor binding pocket (Rogers, G. N., Paulson, J.C., Daniels, R.S., Skehel, J.J., Wilson, I.A., and Wiley, D.C. (1983) Nature 304, 76-78). In this report, the selection in the reverse direction was accomplished starting with a SA alpha 2,3Gal-specific avian virus, A/duck/Ukraine/1/63 (H3N7), yielding SA alpha 2,6Gal-specific variants that exhibit the receptor binding properties characteristic of the human isolates. Selection was again mediated at residue 226 of the hemagglutinin, in this case changing from Gln in the parent virus to Leu in the variants. Although the SA alpha 2,6Gal-specific avian virus variants were stable to passage in MDCK cells, they exhibited dramatic reversion to the SA alpha 2,3Gal-specific phenotype of the parent virus during a single passage in chicken embryos. This was in contrast to the SA alpha 2,6Gal-specific human virus isolates which were stable to passage in both hosts. The reversion of the avian virus variants in eggs provides compelling evidence for host-mediated selection of influenza virus receptor variants.

Salicylic acid-binding protein 2 (SABP2) is essential for the establishment of systemic acquired resistance (SAR) in tobacco; SABP2's methyl salicylate (MeSA) esterase activity is required in healthy systemic tissues of infected plants to release the active defense phytohormone SA from MeSA, which serves as a long-distance signal for SAR. In the current study, we characterize a new gene family from Arabidopsis thaliana encoding 18 potentially active alpha/beta fold hydrolases that share 32-57% identity with SABP2. Of 14 recombinant AtMES (MES for methyl esterase) proteins tested, five showed preference for MeSA as a substrate and displayed SA inhibition of MeSA esterase activity in vitro (AtMES1, -2, -4, -7, and -9). The two genes encoding MeSA esterases with the greatest activity, AtMES1 and -9, as well as AtMES7 were transcriptionally upregulated during infection of Arabidopsis with avirulent Pseudomonas syringae. In addition, conditional expression of AtMES1, -7, or -9 complemented SAR deficiency in SABP2-silenced tobacco, suggesting that these three members of the AtMES family are SABP2 functional homologs (orthologs). Underexpression by knockout mutation and/or RNAi-mediated silencing of multiple AtMES genes, including AtMES1, -2, -7, and -9, compromised SAR in Arabidopsis and correlated with enhanced accumulation of MeSA in the systemic tissue of SAR-induced plants. Together, the data show that several members of the AtMES gene family are functionally homologous to SABP2 and redundant for MeSA hydrolysis and probably SAR. These data suggest that MeSA is a conserved SAR signal in Arabidopsis and tobacco.

alpha-dioxygenases (alpha-DOXs) catalyze the primary oxygenation of fatty acids into a newly identified group of oxylipins. Here we show that expression of the Arabidopsis alpha-DOX1 gene is induced in response to both incompatible and compatible bacterial infections. However, the level of alpha-DOX1 mRNA and dioxygenase activity appears earlier and reaches higher values when infection promotes a hypersensitive reaction. Furthermore, whereas gene expression is confined to necrotic lesions during the hypersensitive response, it occurs throughout the chlorotic area during a compatible interaction. Accumulation of alpha-DOX1 transcripts is impaired in SA-compromised plants and induced by SA and by chemicals generating nitric oxide (NO), intracellular superoxide or singlet oxygen, three signals mediating host cell death. Transgenic plants with altered levels of alpha-dioxygenase react like wild-type plants to a compatible pathogen. In contrast, plants with reduced activity develop a more rapid and severe necrotic response than wild-type plants to incompatible bacteria and paraquat treatment, respectively, and a milder response when alpha-DOX1 is overproduced. Our results suggest that plant alpha-dioxygenases are used to generate lipid-derived molecules for a process that protects plant tissues from oxidative stress and cell death.

The Kruskal-Wallis (KW) nonparametric analysis of variance is often used instead of a standard one-way ANOVA when data are from a suspected non-normal population. The KW omnibus procedure tests for some differences between groups, but provides no specific post hoc pair wise comparisons. This paper provides a SAS(®) macro implementation of a multiple comparison test based on significant Kruskal-Wallis results from the SAS NPAR1WAY procedure. The implementation is designed for up to 20 groups at a user-specified alpha significance level. A Monte-Carlo simulation compared this nonparametric procedure to commonly used parametric multiple comparison tests.

BACKGROUND

Fabry cardiomyopathy is diagnosed by detection of left ventricular hypertrophy (LVH) in patients with alpha-Galactosidase A deficiency. Conventional noninvasive tools are unable to provide a preclinical diagnosis allowing prompt institution of enzymatic therapy.

RESULTS

We studied three groups of patients: 10 patients with causal mutations for Fabry disease and LVH, 10 mutation-positive patients without LV, and 10 healthy relatives without causal mutations and no LVH. All patients with LVH and 6 patients with Fabry disease without LVH with complex repetitive ventricular arrhythmias underwent biventricular endomyocardial biopsy to assess cardiac involvement. In all patients 2-dimensional echocardiography with tissue Doppler analysis in the pulsed Doppler mode was performed: systolic (Sa), early diastolic (Ea), and late diastolic (Aa) velocities were measured, and the Ea/Aa ratio and the dimensionless parameter E/Ea were computed at both corners of the mitral annulus. Histology and electron microscopy studies showed glycosphingolipid deposits in all cases. All mutation-positive patients had significant reduction of Sa, Ea, and Aa velocities at both corners of the mitral annulus compared with normal control subjects. Ea/Aa ratio was significantly lower and E/Ea ratio significantly higher in mutation-positive patients than in control subjects. Patients with LVH showed significantly lower contraction and relaxation tissue Doppler velocities, lower Ea/Aa ratio, and higher E/Ea ratio in comparison with mutation-positive patients with no LVH.

CONCLUSIONS

Fabry cardiomyopathy is characterized by reduced myocardial contraction and relaxation tissue Doppler velocities, detectable even before development of LVH. Tissue Doppler imaging can provide a preclinical diagnosis of Fabry cardiomyopathy, allowing early institution of enzyme replacement therapy.

We have analyzed cell surface-bound carbohydrates in two different model systems for metastasis composed of closely related tumor cell lines with differing metastatic potential. The first system studied was that of the DBA/2-derived T-lymphoma lines (Eb/ESb) and some recently established sublines of ESb with altered metastatic behavior (ESb-M and ESb-MR). The second system consisted of the highly metastatic MDAY-D2 cells, a wheat germ agglutinin-resistant low metastatic subline MDW40, and two metastatic revertants from the latter. The cells were stained with fluorescein isothiocyanate-conjugated lectins and analyzed by flow cytofluorography. All low-metastatic tumor lines expressed receptor sites for the lectins soybean agglutinin (SBA) and Vicia villosa (VV). The metastatic lines had the respective lectin binding sites blocked by sialic acid (SA). A good correlation was found within the cell lineages Eb leads to ESb leads to ESb-M leads to ESb-MR and MDAY-D2 leads to MDW40 leads to MDW40M1 between reactivity of SBA and VV and metastatic potential. The amount of neuraminidase-accessible SA was similar for all cell types (except MDW40) indicating differences in the positioning of SA. For high-metastatic ESb cells, the sialylation of SBA and VV receptor sites was paralleled by a relative decrease of SA associated with receptor sites for peanut agglutinin. Low-metastatic Eb cells, in contrast, had their peanut agglutinin receptor sites sialylated but expressed asialylated SBA and VV receptor sites. Eb cells were also found to have 2-fold higher activities in galactose-specific sialyltransferases. It is proposed that the differences in positioning of SA on the cell surface leading to masking or unmasking of terminal sugars could influence the metastatic potential of tumor cells.

BACKGROUND

Half a century after the inception of the term "successful aging (SA)," a consensus definition has not emerged. The current study aims to provide a comprehensive snapshot of operational definitions of SA.

METHODS

A systematic review across MedLine, PsycInfo, CINAHL, EMBASE, and ISI Web of Knowledge of quantitative operational definitions of SA was conducted.

RESULTS

Of the 105 operational definitions, across 84 included studies using unique models, 92.4% (97) included physiological constructs (e.g. physical functioning), 49.5% (52) engagement constructs (e.g. involvement in voluntary work), 48.6% (51) well-being constructs (e.g. life satisfaction), 25.7% (27) personal resources (e.g. resilience), and 5.7% (6) extrinsic factors (e.g. finances). Thirty-four definitions consisted of a single construct, 28 of two constructs, 27 of three constructs, 13 of four constructs, and two of five constructs. The operational definitions utilized in the included studies identify between <1% and >90% of study participants as successfully aging.

CONCLUSIONS

The heterogeneity of these results strongly suggests the multidimensionality of SA and the difficulty in categorizing usual versus successful aging. Although the majority of operationalizations reveal a biomedical focus, studies increasingly use psychosocial and lay components. Lack of consistency in the definition of SA is a fundamental weakness of SA research.

BACKGROUND

Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs.

RESULTS

Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5'-aminolevulinic acid dehydratase (ALAD, the second step). Similarly, Escherichia coli hemH (FC) deficient strains transformed with human and Wolbachia FC homologues showed significantly different sensitivities to NMMP. This approach enables functional complementation in E. coli heme deficient mutants as an alternative E. coli-based method for drug screening.

CONCLUSIONS

Our studies indicate that the heme biosynthetic genes in the Wolbachia of B. malayi (wBm) might be essential for the filarial host survival. In addition, the results suggest they are likely candidate drug targets based upon significant differences in phylogenetic distance, biochemical properties and sensitivities to heme biosynthesis inhibitors, as compared to their human homologues.

1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned alpha-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-S-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop. 3. SNS and SNS(SA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels. 4. Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I-II loop.

BACKGROUND

A number of sexual and social risk factors for bacterial vaginosis (BV) have been identified. However, many previous studies have used small numbers of patients, or highly selected or convenience samples, or poorly defined populations. This study aims to clarify potential sexual and non-sexual risk factors for BV.

METHODS

Women attending the Sydney Sexual Health Centre with BV, between March 1991 and July 1999, were included. Controls were randomly selected women without BV. Information on the demographics, clinical findings, and sexual and non-sexual risk behaviours were extracted from the clinic database and analysed using SPSS and SAS. A logistic regression model was used to establish which associations with BV persisted.

RESULTS

890 women with BV and 890 controls were studied. Factors that were independently associated with BV were>> or =3 male sexual partners in the past 12 months (OR = 1.60, 95% CI: 1.19 to 2.04), at least one female sexual partner in the past 12 months (OR = 2.1, p = 0.003), a past pregnancy (OR = 1.5, p<0.0006), and smoking. In contrast, women with BV were significantly less likely to have used hormonal contraception (OR = 0.60, 95% CI: 0.51 to 0.81) or to have used condoms consistently (OR = 0.5, 95% CI: 0.31 to 0.71) than controls.

CONCLUSIONS

Our findings may be important for planning a preventive strategy for BV by discouraging smoking and increasing condom use and hormonal contraception among women.

The role of tumor necrosis factor-alpha (TNF-alpha) in human B cell responses was examined and compared with that of interleukin (IL) 1 by assessing the ability of each cytokine to support proliferation and differentiation. Recombinant TNF-alpha (rTNF-alpha) and recombinant IL-1 (rIL-1) each enhanced the generation of immunoglobulin-secreting cells (ISC) in cultures of pokeweed mitogen-stimulated B cells incubated with T cells. To examine the direct effect of rTNF-alpha and rIL-1 on the responding B cell, highly purified peripheral blood B cells were stimulated with Cowan I Staphylococcus aureus (SA). In the absence of T cell factors, proliferation was minimal and there was no generation of ISC. Recombinant IL-2 (rIL-2) supported both responses. Although rTNF-alpha alone did not support SA-stimulated generation of ISC, it did increase SA-stimulated B cell DNA synthesis by two- to eightfold. In addition, rTNF-alpha augmented B cell proliferation in rIL-2 supported SA-stimulated cultures. Moreover, rTNF-alpha enhanced the generation of ISC stimulated by rIL-2 alone or rIL-2 and SA. rIL-1 also augmented DNA synthesis and generation of ISC by B cells stimulated with SA and rIL-2. However, rTNF-alpha enhanced proliferation and ISC generation in SA + rIL-2-stimulated cultures even when they were supplemented with saturating concentrations of rIL-1. Utilizing a two-stage culture system, it was found that the major effect of rTNF-alpha was to enhance responsiveness of SA-activated B cells to rIL-2, whereas it exerted only a minimal effect during initial stimulation. These results indicate that TNF-alpha as well as IL-1 augment B cell responsiveness. Moreover, the ability of rTNF-alpha to enhance B cell responsiveness was not an indirect effect resulting from the induction of Il-1 production by contaminating monocytes, but rather resulted from the delivery of a signal by rTNF-alpha directly to the responding B cell that promoted both proliferation and differentiation after initial activation. The data therefore indicate that human B cell responsiveness can be independently regulated by the action of two separate monocyte-derived cytokines.

TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-alpha converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the complex formed by the catalytic domain of recombinant human TACE and the N-terminal domain of TIMP-3 (N-TIMP-3), and determined its molecular structure with X-ray data to 2.3 A resolution. The structure reveals that TIMP-3 exhibits a fold similar to those of TIMP-1 and TIMP-2, and interacts through its functional binding edge, which consists of the N-terminal segment and other loops, with the active-site cleft of TACE in a manner similar to that of matrix metalloproteinases (MMPs). Therefore, the mechanism of TIMP-3 binding toward TACE is not fundamentally different from that previously elucidated for the MMPs. The Phe34 phenyl side chain situated at the tip of the relatively short sA-sB loop of TIMP-3 extends into a unique hydrophobic groove of the TACE surface, and two Leu residues in the adjacent sC-connector and sE-sF loops are tightly packed in the interface allowing favourable interactions, in agreement with predictions obtained by systematic mutations by Gillian Murphy's group. The combination of favourable functional epitopes together with a considerable flexibility renders TIMP-3 an efficient TACE inhibitor. This structure might provide means to design more efficient TIMP inhibitors of TACE.

Sleep apnea syndrome (SAS), a common disorder, is characterized by repetitive episodes of cessation of breathing during sleep, resulting in hypoxemia and sleep disruption. The consequences of the abnormal breathing during sleep include daytime sleepiness, neurocognitive dysfunction, development of cardiovascular disorders, metabolic dysfunction, and impaired quality of life. There are two types of SAS: obstructive sleep apnea syndrome (OSAS) and central sleep apnea syndrome (CSAS). OSAS is a prevalent disorder in which there is snoring, repetitive apneic episodes, and daytime sleepiness. Anatomical conditions causing upper airway obstruction (obesity or craniofacial abnormalities such as retrognathia or micrognathia) can cause OSAS. CSAS, much less common than OSAS, is a disorder characterized by cessation of breathing which is caused by reduced respiratory drive from the central nervous system to the muscles of respiration. The latter condition is common in patients with heart failure and cerebral neurologic diseases. The diagnosis of SAS requires assessment of subjective symptoms and apneic episodes during sleep documented by polysomnography. Treatments of OSAS include continuous positive airway pressure (CPAP), oral appliances, and surgery; patients with CSAS are treated with oxygen, adaptive servo-ventilation, or CPAP. With assessment and treatment of the SAS, patients usually have resolution of their disabling symptoms, subsequently resulting in improved quality of life.

Salicylic acid (SA) is a phenolic metabolite produced by plants and is known to play an important role in several physiological processes, such as the induction of plant defense responses against pathogen attack. Here, using the Arabidopsis thaliana-Pseudomonas aeruginosa pathosystem, we provide evidence that SA acts directly on the pathogen, down regulating fitness and virulence factor production of the bacteria. Pseudomonas aeruginosa PA14 showed reduced attachment and biofilm formation on the roots of the Arabidopsis mutants lox2 and cpr5-2, which produce elevated amounts of SA, as well as on wild-type Arabidopsis plants primed with exogenous SA, a treatment known to enhance endogenous SA concentration. Salicylic acid at a concentration that did not inhibit PA14 growth was sufficient to significantly affect the ability of the bacteria to attach and form biofilm communities on abiotic surfaces. Furthermore, SA down regulated three known virulence factors of PA14: pyocyanin, protease, and elastase. Interestingly, P. aeruginosa produced more pyocyanin when infiltrated into leaves of the Arabidopsis transgenic line NahG, which accumulates less SA than wild-type plants. This finding suggests that endogenous SA plays a role in down regulating the synthesis and secretion of pyocyanin in vivo. To further test if SA directly affects the virulence of P. aeruginosa, we used the Caenorhabditis elegans-P. aeruginosa infection model. The addition of SA to P. aeruginosa lawns significantly diminished the bacterium's ability to kill the worms, without affecting the accumulation of bacteria inside the nematodes' guts, suggesting that SA negatively affects factors that influence the virulence of P. aeruginosa. We employed microarray technology to identify SA target genes. These analyses showed that SA treatment affected expression of 331 genes. It selectively repressed transcription of exoproteins and other virulence factors, while it had no effect on expression of housekeeping genes. Our results indicate that in addition to its role as a signal molecule in plant defense responses, SA works as an anti-infective compound by affecting the physiology of P. aeruginosa and ultimately attenuating its virulence.

BACKGROUND

Pigs are considered susceptible to influenza A virus infections from different host origins because earlier studies have shown that they have receptors for both avian (sialic acid-alpha-2,3-terminal saccharides (SA-alpha-2,3)) and swine/human (SA-alpha-2,6) influenza viruses in the upper respiratory tract. Furthermore, experimental and natural infections in pigs have been reported with influenza A virus from avian and human sources.

METHODS

This study investigated the receptor distribution in the entire respiratory tract of pigs using specific lectins Maackia Amurensis (MAA) I, and II, and Sambucus Nigra (SNA). Furthermore, the predilection sites of swine influenza virus (SIV) subtypes H1N1 and H1N2 as well as avian influenza virus (AIV) subtype H4N6 were investigated in the respiratory tract of experimentally infected pigs using immunohistochemical methods.

RESULTS

SIV antigen was widely distributed in bronchi, but was also present in epithelial cells of the nose, trachea, bronchioles, and alveolar type I and II epithelial cells in severely affected animals. AIV was found in the lower respiratory tract, especially in alveolar type II epithelial cells and occasionally in bronchiolar epithelial cells. SA-alpha-2,6 was the predominant receptor in all areas of the respiratory tract with an average of 80-100% lining at the epithelial cells. On the contrary, the SA-alpha-2,3 was not present (0%) at epithelial cells of nose, trachea, and most bronchi, but was found in small amounts in bronchioles, and in alveoli reaching an average of 20-40% at the epithelial cells. Interestingly, the receptor expression of both SA-alpha-2,3 and 2,6 was markedly diminished in influenza infected areas compared to non-infected areas.

CONCLUSIONS

A difference in predilection sites between SIV and AIV virus was found, and this difference was in accordance with the distribution of the SA-alpha-2,6 and SA-alpha-2,3 receptor, respectively. The results indicated that the distribution of influenza A virus receptors in pigs are similar to that of humans and therefore challenge the theory that the pig acts as a mixing vessel between human and avian influenza viruses. Furthermore, it was shown that AIV prefers to infect alveolar type II epithelial cells in pigs. This corresponds with findings in humans emphasising the resemblance between the two species.

It has previously been shown that the M (E1) glycoprotein of mouse hepatitis virus strain AAA detailed pulse-chase analysis was made of the addition of O-linked sugars to the M protein; upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three different electrophoretic forms could be distinguished that corresponded to the sequential acquisition of N-acetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (SA). A fourth and fifth form could also be detected which we were unable to identify. Following Brefeldin A treatment, the M protein still acquired GalNAc, Gal, and SA, but the fourth and fifth forms were absent, suggesting that these modifications occur in the trans-Golgi network (TGN). In contrast, in the presence of BFA, the G protein of vesicular stomatitis virus (VSV), which contains N-linked oligosaccharides, acquired Gal and fucose but not SA. These results are consistent with earlier published data showing that Golgi compartments proximal to the TGN, but not the TGN itself, relocate to the endoplasmatic reticulum/intermediate compartment. More importantly, our data argue that, whereas addition of SA to N-linked sugars occurs in the TGN the acquisition of both SA on O-linked sugars and the addition of fucose to N-linked oligosaccharides must occur in Golgi compartments proximal to the TGN. The glycosylation of the M protein moreover indicates that it is transported to trans-Golgi and TGN. This was confirmed by electron microscopy immunocytochemistry, showing that the protein is targeted to cisternae on the trans side of the Golgi and co-localizes, at least in part, with TGN 38, a marker of the TGN, as well as with a lectin specific for sialic acid.

OBJECTIVE

Recent evidence shows that calcium released from the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart rate. The aim of this study was to investigate the subcellular distribution of ryanodine receptors in the guinea-pig sino-atrial (SA) node and to determine their functional role in the regulation of pacemaker frequency in response to beta-adrenoceptor stimulation.

METHODS

Monoclonal antibodies raised against the cardiac ryanodine receptor were used with confocal microscopy to investigate ryanodine receptor distribution in single guinea-pig SA node cells. The functional role of ryanodine receptors was investigated in both multicellular SA node/atrial preparations and in single SA node cells.

RESULTS

Ryanodine receptor labelling was observed in all SA node cells studied and showed both subsarcolemmal and intracellular staining. In the latter, labelling appeared as transverse bands with a regular periodicity of approximately 2 microm. This interval resembled that of the expected sarcomere spacing but did not, however, depend on the presence of transverse tubules. The bands of ryanodine receptors appeared to be located in the region of the Z lines, based on co-distribution studies with antibodies to alpha-actinin, myomesin and binding sites for phalloidin. Functional studies on single SA node cells showed that application of ryanodine (2 micromol/l) reduced the rate of firing of spontaneous action potentials (measured using the perforated patch clamp technique) and this was associated with changes in action potential characteristics. Ryanodine also significantly decreased the positive chronotropic actions of isoprenaline in both multicellular and single cell preparations. In single cells exposed to 100 nmol/l isoprenaline, ryanodine caused a decrease in the rate of firing and this was associated with a decrease in the amplitude of the measured calcium transients.

CONCLUSIONS

These findings are the first to show immunocytochemical evidence for the presence and organisation of ryanodine receptor calcium release channels in mammalian SA node cells. This study also provides evidence of a role for ryanodine sensitive sites in the beta-adrenergic modulation of heart rate in this species.

It is well known that abscisic acid (ABA) antagonizes gibberellin (GA)-promoted seed germination. Recent circumstantial evidence suggests that salicylic acid (SA) also inhibits seed germination in maize and Arabidopsis. Our study shows that SA blocks barley seed germination in a dosage dependent manner. As an initial effort to addressing the mechanism controlling the crosstalk of SA, GA and ABA signaling in barley, we studied the regulation of alpha-amylases by SA and a WRKY gene whose expression is modulated by these hormones. Assays of alpha-amylase activity reveal that GA-induced alpha-amylase production in aleurone cells is inhibited by bioactive SA, but not its analogs, 3-hydroxybenzoic acid and 4-hydroxybenzoic acid. This inhibitory effect is unlikely due to repressing alpha-amylase secretion or inhibiting alpha-amylase enzyme activities. Northern blot analyses indicate that SA suppresses GA-induced expression of a barley low pI alpha-amylase gene (Amy32b). Because our previous data indicate that ABA-inducible and GA-suppressible WRKY genes inhibit the expression of alpha-amylase genes in rice, we studied the steady state mRNA levels of a barley WRKY gene, HvWRKY38. The expression of HvWRKY38 in barley aleurone cells is down-regulated by GA, but up-regulated by SA and ABA. However, the regulation of HvWRKY38 by SA appears to be different from that of ABA in term of the kinetics and levels of induction. Over-expression of HvWRKY38 in aleurone cells by particle bombardment blocks GA induction of the Amy32b promoter reporter construct (Amy32b-GUS). Therefore, HvWRKY38 might serve as a converging node of SA and ABA signal pathways involved in suppressing GA-induced seed germination.

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. The present study was undertaken to investigate whether treatment with the antifibrotic drug pirfenidone attenuates the bleomycin (BL)-induced overexpression of HSP47 in the lungs. Male ICR mice were intravenously injected with BL or saline (SA). Pirfenidone or control drug (CD) was administered 14 days after commencement of BL or SA, and continued throughout the course of the experiment. The mice were randomly divided into three experimental groups: 1) SA-treated with CD (SA group); 2) BL-treated with CD (BL group); and 3) BL-treated with pirfenidone (pirfenidone group). Lungs of the pirfenidone group showed a marked reduction of fibrotic lesions compared with the corresponding BL group. Immunohistochemical studies showed that BL treatment significantly increased the number of macrophages, myofibroblasts, HSP47-positive type II pneumocytes and HSP47-positive interstitial spindle-shaped cells. Treatment with pirfenidone significantly reduced the number of these cells compared with the corresponding BL group. Furthermore, treatment with pirfenidone significantly suppressed the BL-induced increase of the positive ratio of HSP47 and alpha-smooth muscle actin to interstitial spindle-shaped cells. The present study results showed that pirfenidone inhibited heat shock protein 47-positive cells and myofibroblasts, the principal cells responsible for the accumulation and deposition of extracellular matrix seen in pulmonary fibrosis.

To compare features of the receptor-binding sites (RBSs) of different influenza virus hemagglutinins (HA), binding of a number of synthetic sialic acid (SA) analogs and natural sialosides by a panel of about 30 human influenza A and B virus strains was studied in a competitive ligand binding assay. For all the viruses tested, the N-acetyl group of Neu5Ac, as well as the natural orientation of the carboxylic group at C2 and the hydroxylic group at C4, was essential for binding. Significant type- and subtype-specific differences were observed in virus recognition of asialic parts of sialosides. H1 strains, unlike H3 and type B viruses, were found to bind alpha 2-6-sialyl-N-acetyllactosamine with about an order of magnitude higher affinity than alpha 2-6-sialyllactose (6'SL). The H1 viruses and the H3 strains with Gln in position 226 of HA, but not the H3 strains with Leu-226, bound 6'SL with a lower affinity than alpha 2-3-sialyllactose; this effect correlated clearly with the preferential binding by the former viruses of unsubstituted alpha Neu5Ac compared to methyl alpha-glycoside of Neu5Ac. Thus, differentiation between the types of the SA-Gal linkage by the A viruses appeared to depend, at least partially, upon the recognition by the HA of the first hydrocarbon group of the aglycon. Type B virus strains were distinct in having a lower affinity for the Neu5Ac moiety and in providing a higher contribution of the asialic portions of sialosides to the HA-ligand interactions. The last effects are presumably due to the amino acid insertions in the type B HA surrounding the RBS, which makes the receptor-binding pocket deeper. The results obtained in the present investigation indicate that while the functional groups of Neu5Ac studied are recognized by the RBSs of all influenza viruses, the magnitude of their contribution to the binding energy, as well as the contribution of the asialic portion of the receptor, may vary in dependence upon the virus type, subtype, and strain.

Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare, Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such SA-mediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.

Two hundred seven patients with DSM-IV Pathological Gambling Disorder completed both the Gambling Symptom Assessment Scale (G-SAS) and the Yale-Brown Obsessive-Compulsive Scale--modified for Pathological Gambling (PG-YBOCS) at baseline visit and weekly or biweekly thereafter during the 12-week study period. The week 1 to week 2 visit data were used to assess test-retest reliability. Weekly or biweekly data were used for the G-SAS validity. The PG-YBOCS reliability and validity data have been published previously. We used the PG-YBOCS as the established scale and compared the G-SAS performance with the PG-YBOCS. Test-retest reliability was statistically significant. The correlations between the G-SAS and the PG-YBOCS and Clinical Global Impression rating were excellent. Findings suggest that the G-SAS is reliable and valid in assessing changes in symptoms during a drug treatment study.

Salivary proteins and glycoproteins that participate in the formation of 2-h in vivo enamel pellicle were determined utilizing polyacrylamide gel electrophoresis [sodium dodecyl sulphate (SDS)-PAGE and anionic PAGE]/Western transfer analyses, and specific radiolabelling/SDS-PAGE fluorography. The sensitivity of these methods permitted the identification of individual members of different salivary protein families. The major components of this pellicle were salivary alpha-amylase, cysteine-containing phosphoprotein (CCP or cystatins), salivary mucin and sIgA. Glycosylated amylase was present in larger quantity than the non-glycosylated species. Only CCP1 (cystatin SA-I) of the cysteine-containing phosphoprotein family was identified. The higher molecular-weight salivary mucin (MG1), but not the lower molecular-weight species (MG2), was detected. These results extend earlier observations regarding the selective nature of salivary protein adsorption to enamel surface by demonstrating that only specific members of salivary protein families are involved in 2-h in vivo enamel pellicle formation. The findings also suggest that individual family members may have different functions in the mouth.