Selective, rapid and simultaneous determination of ergosterol and ergocalciferol in mushrooms by UPLC-Q-TOF-MS.

An ultra-performance liquid chromatography coupled to atmospheric pressure chemical ionization-quadrupole time-of-flight mass spectrometry method has been optimized and validated for the determination of ergosterol and ergocalciferol in mushroom samples, using cholecalciferol as surrogate standard. The separation was carried out with a Synergi Hydro-RP column (100 mm x 3.00 mm i.d, 2.5 µm particle size), (Phenomenex, CA, USA) column, thermostated at 35 °C. The mobile phase was 0.1 % formic acid aqueous solution and methanol in gradient elution mode and it was achieved in 5 min approximately. Detection was achieved by atmospheric pressure chemical ionization in positive mode and quadrupole time-of-flight mass spectrometry. Desolvation and interface temperatures were set at 500 °C and 150 °C, respectively. The recoveries obtained were within 92-105 % for ergosterol, 77-81 % for ergocalciferol and 83-87 % for cholecalciferol. Method limits of detection were 0.4 and 0.5 µg g-1 for ergosterol and ergocalciferol, respectively, and method limits of quantitation were 1.2 and 1.3 µg g-1 for ergosterol and ergocalciferol, respectively. A rapid and simple extraction procedure using small amount of sample (100 mg) with hexane was optimized and the method was applied to the determination of ergosterol and ergocalciferol in white button mushrooms (Agaricus bisporus var. bisporus) exposed to UV irradiation. Results were compared to the corresponding non-irradiated mushrooms.

Patient-provider relationship and perceived provider weight bias among American Indians and Alaska Natives.

Objective: The objective of this study was to examine patient-provider relationships among American Indians and Alaska Native (AI/AN) patients by examining associations between patient activation, perceived provider weight bias and working alliance. Patient activation is generally defined as having the knowledge, skills and confidence to manage one's health. Methods: Among a sample of 87 AI/AN adults presenting for general medical care at an urban clinic in the north-west region of the USA, ordinary least squares regression analysis was completed to examine associations. Results: Better working alliance scores were associated with increased patient activation, while perceived provider weight bias was associated with reduced patient activation. In addition, those with class II obesity had decreased patient activation. Conclusion: These findings point to the importance of a positive patient-provider relationship in AI/ANs. Optimal patient engagement and subsequent health outcomes warrant additional consideration of patients' perceptions of provider weight bias within the context of health promotion and interventions.

The combined effects of pyridostigmine and chronic stress on brain cortical and blood acetylcholinesterase, corticosterone, prolactin and alternation performance in rats.

Thousands of soldiers who served in the Gulf War have symptoms that have been collectively termed Gulf War Illness (GWI). It has been suggested that a combination of operational stress and pyridostigmine, a drug given as a pretreatment to protect soldiers against the effects of exposure to nerve agents, might have had unexpected adverse health effects causing these symptoms. Our laboratory has previously modeled operational stress in rats using a paradigm of around-the-clock intermittent signalled footshock. In the present studies, this model was used to investigate the potential synergistic effects of chronic stress and pyridostigmine on physiology and behavior. Seventy-two rats were trained to perform an alternation lever pressing task to earn their entire daily food intake. The rats were then implanted with osmotic minipumps containing vehicle, pyridostigmine (25 mg/ml pyridostigmine bromide) or physostigmine (20 mg/ml eserine hemisulfate). The pumps delivered 1 microl/h, which resulted in a cumulative dosing of approximately 1.5 mg/kg/day of pyridostigmine or 1.2 mg/kg/day of physostigmine, equimolar doses of the two drugs. The rats were then returned to their home cages where performance continued to be measured 24 h/day. After 4 days, 24 of the 72 rats were trained to escape signalled footshock (avoidance-escape group) and 24 other rats (yoked-stressed group) were each paired to a rat in the avoidance-escape group. The remaining 24 rats were not subjected to footshock (unstressed group). Shock trials were intermittently presented in the home cage 24 h/day for 3 days, while alternation performance continued to be measured. Since only 12 test cages were available, each condition was repeated to achieve a final n of six rats per group. Pyridostigmine and physostigmine each decreased blood acetylcholinesterase levels by approximately 50%. Physostigmine also decreased brain cortical acetylcholinesterase levels by approximately 50%, while pyridostigmine had no effect on cortical acetylcholinesterase activity. Alternation performance was impaired on the first day of stress and then recovered. Neither pyridostigmine nor physostigmine affected performance in the absence of stress or increased the effects of stress alone. Corticosterone was significantly increased in the yoked stress group compared to unstressed controls. These data suggest that pyridostigmine does not exacerbate the effects of stress on performance or levels of stress hormones. Furthermore, these data do not suggest that stress enables pyridostigmine to cross the blood brain barrier.

2-methoxyestradiol blocks cell-cycle progression at G(2)/M phase and inhibits growth of human prostate cancer cells.

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17beta-estradiol, is present in human blood and urine. Here we show for the first time that 2-ME significantly inhibited the growth of normal prostate epithelial cells and androgen-dependent LNCaP and androgen-independent DU145 prostate cancer cells. This growth inhibition was accompanied by a twofold increase in the G(2)/M population, with a concomitant decrease in the G(1) population, as shown by cell-cycle analysis. 2-ME treatment affected the cell-cycle progression of prostate cancer cells specifically by blocking cells in the G(2) phase. Immunoblot analysis of the key cell-cycle regulatory proteins in the G(2)/M phase showed a 14-fold increase in the expression of p21 and an eightfold increase in the expression of p34 cell division cycle 2 (cdc2). We also found an accumulation of phosphorylated cdc2 after 2-ME treatment. Furthermore, Wee 1 kinase was detectable after 2-ME treatment. 2-ME treatment also led to an increase in the activity of caspase-3, followed by apoptosis, as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end-labeling and fluorescein isothiocyanate-poly(ADP-ribose) polymerase assay. Estrogen receptor levels did not change after treatment with 2-ME. Examination of the signaling pathways that mediate 2-ME-induced apoptosis showed reduction in the level of p53 expression and its DNA-binding activity. Given the fact that p53 mutations are common in patients with metastatic prostate cancer, our finding that 2-ME-mediated growth inhibition of human prostate cancer cells occurred in a p53-independent manner has considerable clinical significance. These findings, combined with the limited toxicity of 2-ME, may have significant implications for alternative treatment of advanced prostate cancer.

NF-kappaB-dependent fractalkine induction in rat aortic endothelial cells stimulated by IL-1beta, TNF-alpha, and LPS.

Fractalkine is an endothelial cell-derived CX3C chemokine that is chemotactic mainly to mononuclear cells. Fractalkine was induced in rat aortic endothelial cells (RAEC) by interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) transcriptionally and translationally. This induction correlated with increased NF-kappaB DNA binding activity as determined by gel mobility shift assay. Supershift assays revealed that the NF-kappaB subunits p50 and p65 were responsible for kappaB binding. Accordingly, we examined the role of NF-kappaB in fractalkine induction in RAEC through the use of an adenovirus-mediated mutant IkappaB as a specific inhibitor. Delivery of a dominant-negative form of IkappaBalpha in RAEC dramatically reduced the induction of fractalkine by these stimuli, suggesting a role for NF-kappaB activation in fractalkine induction. The inhibition of fractalkine expression by two potent NF-kappaB inhibitors, sulfasalazine and sanguinarine, further supported the central role of NF-kappaB in fractalkine transcription regulation and suggested a novel therapeutic target aimed at modulating leukocyte endothelial cell interaction.

Heparin-binding EGF-like growth factor contributes to reduced glomerular filtration rate during glomerulonephritis in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti- glomerular basement membrane (anti-GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti-HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations.

Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro.

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76-77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse-phase high-performance liquid chromatography (RP-HPLC) method was used to measure digested peptides. A dose as high as 600 microM of substance P, and 11-amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B-I, QF site 24-25) is 39 amino acids in length, and sequence comparison of B-I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer-aided secondary structure computations predict alpha-helical structures flanking the QF site for VAMP2 and for the upstream sequence of B-I. Although predictions for the downstream sequence give nearly equal tendencies for alpha-helical and beta-sheet structures, Yi et al. showed that the downstream sequence is likely to be the alpha-helix based on their examination of buforin II (B-II, a 21-amino acid subset of B-I (16-36)), which includes the QF site and the downstream sequence of B-I. Buforin I was found not to be a substrate for BoNT/B; however, B-I dose dependently and competitively inhibited BoNT/B activity, yielding IC(50) = 1 x 10(-6) M. In contrast, B-II was not a substrate for BoNT/B and exhibited only 25% of the B-I inhibition of BoNT/B. Two additional B-I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B-I amino acids 1-36) and peptide 24 (24 mer; B-I amino acids 16-39). Peptide 24 had a similar inhibitory effect to B-II (ca. 25% of B-I) but peptide 36 was almost 50% as potent as B-I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.

Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication.

BACKGROUND: Fractalkine is a newly identified T-cell and monocyte/macrophage (Mphi) chemokine with a transmembrane domain and is a cell-surface protein on activated endothelium. It can mediate adhesion of cells expressing the fractalkine receptor CX3CR1. These unique features make fractalkine well suited for leukocyte recruitment in tissues with high blood flow as in the renal glomerulus. METHODS: Fractalkine expression in glomeruli and response of isolated glomerular inflammatory cells to fractalkine were studied in the Wistar-Kyoto (WKY) crescentic glomerulonephritis model. Antibody was used to confirm the proinflammatory role of fractalkine. RESULTS: Fractalkine was markedly induced in the endothelium of nephritic rat glomeruli, and inflammatory leukocytes infiltrating the glomeruli expressed increased levels of CX3CR1. Anti-CX3CR1 antibody treatment dramatically blocked leukocyte infiltration in the glomeruli, prevented crescent formation, and improved renal function. CONCLUSIONS: Fractalkine plays a central role in leukocyte trafficking at the endothelium in the high-flow glomerular circuit and, in turn, implicates CX3CR1 as a prime drug target for therapeutic intervention of endothelium-related inflammatory diseases.

In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II.

Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.

Xanthurenic acid induces gametogenesis in Plasmodium, the malaria parasite.

A small, heat stable chromophore extracted from mosquitoes has recently been implicated as the signal that induces mating of Plasmodium, the malaria parasite. We have used high resolution electrospray mass spectrometry to determine that this gamete activation factor (GAF) has a m/z = 205.0450, suggesting a molecular species composition of C10H7NO4. Xanthurenic acid (XA), a product of tryptophan catabolism, was determined to have an elemental composition, ultraviolet absorbance maxima, and mass spectrum consistent with those characteristics of GAF. XA activated gametogenesis of Plasmodium gallinaceum and P. falciparum in vitro at concentrations lower than 0.5 microM in saline buffered to pH 7.4. A structural analog of XA, kynurenic acid (C10H6NO3), also activated gametogenesis but only at higher concentrations and with less effect. We propose that XA is GAF. This is the first evidence that XA has induction activity.

Effect of reduction of nitric oxide on plasma and kidney tissue angiotensin II levels.

Nitric oxide synthase (NOS) blockade increases blood pressure (BP) and modifies glomerular and tubular function. Angiotensin II (AII) blockade restores glomerular and tubular function but does not lower BP. We measured plasma renin activity (PRA), plasma (AIIp), and kidney tissue (AIIk) AII with radioimmunoassay to investigate the dissociation between renal and systemic effects of NOS blockade. Two period clearance studies followed by plasma and renal tissue harvesting were performed in seven groups of rats. Groups 1 and 1A served as controls. Groups 2 and 2A received NaCl-NaHCO3 during the first period and N(G)-monomethyl-L-arginine (L-NMMA, 0.5 mg/kg/min) during the second period. Group 3 was similar to group 2 but renal perfusion pressure (RPP) was maintained constant by using an aortic snare. Groups 4 and 4A received N(G)-nitro-L-arginine-methyl ester (L-NAME, 5 mg/100 mL of drinking water) for 2 weeks. NOS blockers decreased AIIp (group 1, 74 +/- 7 pg/mL; group 2, 22 +/- 1 pg/mL; group 3, 26 +/- 1 pg/mL; group 4, 19 +/- 3 pg/mL). The decrease in AIIp was a direct effect of L-NMMA independent of changes in perfusion pressure, as AIIp was similar in group 3 (normal RPP) and groups 2 and 4 (increased RPP). Measurements of PRA and AIIp demonstrated a similar reduction in PRA and AIIp in rats treated with NOS blocker. Although NOS blockers decreased AIIp, acute or chronic administration of NOS blockers did not modify AIIk (group 1, 1,192 +/- 51; group 2, 1,354 +/- 85; group 3, 1,348 +/- 180; group 4, 1,276 +/- 172 pg/kidney). Our findings demonstrate that NO blockers produce a dissociation between plasma and kidney AII levels. This dissociation can explain the beneficial effects of AII blockers on renal function and their lack of antihypertensive effects in anesthetized rats treated with NOS blockers.

Neuroprotective role of c-fos antisense oligonucleotide: in vitro and in vivo studies.

We investigated the dose-response and time-course of c-fos antisense oligodeoxynucleotide (ASO) treatment against excitatory amino acid (EAA)-induced neurotoxicity in rat hippocampal neurons. Glutamate (in vitro) or NMDA (in vivo) produced significant neuronal degeneration. Neuroprotection produced by 30 min or 4 h pretreatment with c-fos ASO in cultured hippocampal neurons was dose-dependent. In vivo, bilateral intrahippocampal injections of c-fos ASO (0.025 nmol/site) was neuroprotective when administered 30 min before or after NMDA treatment. However, 4 h pretreatment was ineffective. A higher dose (0.125 nmol) of c-fos ASO was neurotoxic and failed to afford neuroprotection regardless of the treatment schedule. Collectively, these results demonstrate a neuroprotective effect of c-fos ASO against EAA-induced neuronal injury supporting a causative role of c-fos expression in EAA neurotoxicity.

Isolation of a substance from the mosquito that activates Plasmodium fertilization.

We have isolated a small, heat stabile, hydrophilic molecule from the gut lumen of unfed, female Anopheles stephensi that is a potent inducer of gametogenesis in Plasmodium falciparum and P. gallinaceum at a hydrogen ion concentration, pH 7.4, that normally suppresses activation. This gamete activation factor (GAF) was purified using reverse phase high performance liquid chromatography and determined to have a major ion m/z of 206.1 by low resolution electrospray mass spectrometry. The molecule, which was also found in the heads of both female and male A. stephensi, absorbed light in the ultraviolet region at three maxima (lambda(max) = 213, 245 and 350 nm); the 245/350 nm absorbance ratio was 7.0. The structure of the molecule and its normal function in the mosquito are not yet known, but in a sample of diverse insect species, extracts from those that feed on blood were bioactive. We propose that GAF is the previously observed malaria exflagellation factor (MEF).

Apoptosis of L1210 Leukemia Cells Induced by 3-Deazaadenosine Analogs: Differential Expression of c-myc, NF-Kappa B and Molecular Events.

A new class of potent apogens (apoptosis-inducing agents) has been identified, consisting of 3-deazaadenosine (DZA), 3-deaza-(+/-)aristeromycin (DZAri) and 1-beta-D-arabinofuranosyl-1H-imidazo[4,5-&cumacr;]pyridine (ara-3-deazaadenine; DZAra-A). They are inhibitors of S-adenosylhomocysteine hydrolase and indirect inhibitors of methylation. Furthermore, they have also been found to form 3-deaza-nucleotide analogs. The DZA analogs, DZA, DZAri, and DZAra-A, induced DNA fragmentation in a dose- and time-dependent manner, reaching a maximum at 250 &mgr;M after 72 h. Cycloheximide at 0.5 &mgr;g/ml completely blocked the DNA fragmentation induced by 250 &mgr;M of each of the analogs. Interestingly, exogenous 100 &mgr;M L-homocysteine thiolactone abrogated the DNA fragmentation caused by DZAri and DZAra-A, but not by DZA. Flow cytometric analysis showed that DZA arrested the cells in the G(2)/M phase, whereas the S phase was arrested by DZAri. Correlated with the effect of DZA was a rapid decrease in the expression of c-myc, whereas nur77 and GAPDH were unaffected. In comparison, there was an elevated expression of IFN-gamma mRNA without apparent change in bax, p53 or GAPDH mRNA after 24 h. After treatment with DZA, there was an elevated expression of NF-kappaB DNA binding activity, which became more pronounced at 24 h. Simultaneously, there was an apparent disappearance of AP-1 activity. Thus, DZA most likely inhibited the RNA synthesis of c-myc, a reduction of which could trigger a cascade of gene transcription leading to apoptosis in L1210 cells. Copyright 1997 S. Karger AG, Basel

Effect of angiotensin II on the renal response to amino acid in rats.

Administration of the nitric oxide (NO) synthase blocker, N(G)-monomethyl L-arginine prevents the increase in glomerular filtration rate (GFR) normally observed with glycine, an effect that is restored by angiotensin II (AII) blockers. These findings suggest that changes in NO and AII dictate the presence or absence of renal vasodilation during amino acid (AA) infusion. We examined the effect of branched-chain (BCAA) and non-branched-chain (NBCAA) AA on GFR, NO, and AII to determine if abnormal NO or AII responses could explain the absence of vasodilation with BCAA. Our findings demonstrated that NBCAA increased GFR and NO and did not modify AII, either plasma (AIIp) or kidney (AIIk) AII. The response with BCAA was strikingly different. L-Valine increased GFR without modifying NO or AII. L-Leucine increased AIIk and NO but did not increase GFR. Administration of AII blockers (captopril or losartan) was associated with an increase in GFR during infusion of leucine. Single nephron studies demonstrated that increased AIIk with L-leucine was associated with decreased absolute proximal reabsorption and probably activation of the tubuloglomerular feedback. An AA-specific increase in AIIk is critical to inhibition of the normal renal response to AA infusion. NO generation is an important mediator but not the sole mechanism that determines the increase in GFR during amino acid infusion.

Management of ocular emergencies and urgent eye problems.

Evaluation of the patient with an acute eye problem begins with documentation of the level of vision in each eye, except in the case of a splash injury. In such cases, immediate copious irrigation is of critical importance. Subconjunctival hemorrhage is common and, typically, completely benign. Herpes simplex infection is painful and can lead to extensive damage. Herpes zoster infection is usually accompanied by skin lesions and can be effectively treated with oral acyclovir or famcyclovir. In patients with Bell's palsy, the eye must be carefully protected to prevent secondary injury. Corneal abrasions heal rapidly when antibiotics and patch protection are provided. Acute infections of the eyelids and conjunctivae usually respond well to topical antibiotics and warm compresses. Traumatic injuries require careful evaluation and, frequently, referral to an ophthalmologist.

Role of angiotensin in the regulation of renal response to proteins.

Intrarenal and extrarenal humoral factors have been proposed as mediators and modulators of the renal hyperemic response to amino acid infusion. Among the potential modulators, angiotensin II (AII) constitutes the most important candidate due to its critical role in the control of glomerular and tubular function. The modulatory effect of AII has been assessed by (1) measuring the changes in plasma renin activity (PRA)/AII during the normal hyperemic response, and (2) by assessing the levels of PRA/AII and the response to AII-suppressing agents in conditions with no vasodilatory response during amino acid infusion. Administration of a protein load in normal animals or humans does not modify PRA/AII. Absence of a vasodilatory response in various experimental conditions (nitric oxide blockade in normal rats, experimental models of hypertension, diabetes mellitus, chronic glomerulonephritis, cyclosporine administration) is characterized by a significant decrease in proximal tubular reabsorption during amino acid infusion. Converting enzyme inhibitors or AII receptor antagonist restore normal tubular function and the increase in glomerular filtration rate during amino acid infusion. Absence of a vasodilatory response is also associated with increases in kidney AII levels in some of these conditions. These results suggest that (1) AII modulates the amino acid-induced hyperemia through its inhibitory effect on proximal tubular reabsorption and activation of the tubuloglomerular feedback system, and (2) that the expression of the modulatory effect of AII may depend on the interaction between AII and other intrarenal systems like nitric oxide.

Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide.

Reactive oxygen intermediates (ROIs) play an important role in inflammatory processes as mediators of injury and potentially in signal transduction leading to gene expression. Cyclooxygenase (COX) is a rate-limiting enzyme in prostanoid biosynthesis, and its recently cloned inducible form, COX-2, is induced by proinflammatory cytokines. This study linked ROIs to the signaling pathways that induce COX-2 expression. The hydroxyl radical scavengers DMSO (1%), as well as di- and tetramethylthiourea, inhibited IL-1-, TNF alpha-, and LPS-induced COX-2 expression in rat mesangial cells. The suppression of COX-2 mRNA expression correlated with the COX-2 protein level. In comparison with the prolonged induction of the inducible gene encoding protein-tyrosine phosphatase by hydrogen peroxide, the COX-2 gene was only transiently induced. Protein-tyrosine phosphatase is also induced by heat shock and chemical stress, whereas COX-2 is not. Superoxide was a more potent inducer for COX-2 than hydrogen peroxide. In addition, NADPH stimulated COX-2 expression, and an inhibitor of NADPH oxidase blocked COX-2 expression induced by TNF alpha. COX-2 and KC gene expression costimulated by IL-1 were inhibited differentially by the scavengers. These studies demonstrate that oxidant stress is a specific and important inducer of COX-2 gene expression. This induction may contribute to the deleterious amplification of prostanoids in inflammation and compound the direct effects of ROI production.

Anti-human immunodeficiency virus 1 (HIV-1) activities of 3-deazaadenosine analogs: increased potency against 3'-azido-3'-deoxythymidine-resistant HIV-1 strains.

3-Deazaadenosine (DZA), 3-deaza-(+/-)-aristeromycin (DZAri), and 3-deazaneplanocin A (DZNep) are powerful modulators of cellular processes. When tested against H9 cells infected acutely with two different strains of human immunodeficiency virus 1 (HIV-1) and in the chronically infected monocytoid cell lines U1 and THP-1, the 3-deazanucleosides caused a marked reduction in p24 antigen production. Similar reductions in p24 antigen were seen in phytohemagglutinin-stimulated peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Strikingly, in comparing the therapeutic indices between the paired pre- and post-3'-azido-3'-deoxythymidine (AZT) treatment HIV-1 isolates, DZNep and neplanocin A showed an increase of 3- to 18-fold in their potency against AZT-resistant HIV-1 isolates. In H9 cells treated with DZNep and DZAri, the formation of triphosphate nucleotides of DZNep and DZAri was observed. The mode of action of DZNep and DZAri appears complex, at least in part, at the level of infectivity as shown by decreases in syncytia formation in HIV-1-infected H9 cells and at the level of transcription as both drugs inhibited the expression of basal or tat-induced HIV-1 long terminal repeat chloramphenicol acetyltransferase activity in stably transfected cell lines. Since DZNep induced in H9 cells a rapid expression of nuclear binding factors that recognize the AP-1 transcription site, the anti-HIV-1 activity of the DZA analogs could partly be the induction of critical factors in the host cells. Thus, the 3-deazanucleoside drugs belong to an unusual class of anti-HIV-1 drugs, which may have therapeutic potential, in particular against AZT-resistant strains.