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PubMed2005

Pre-junctional effects of oximes on [3H]-acetylcholine release in rat hippocampal slices during soman intoxication.

In this study, the non-reactivating effects of oximes in the hippocampus of the rat are investigated. The potassium (51 mM) evoked release of [(3)H]-acetylcholine and the liberation of [(3)H]-choline were determined in hippocampal slices following in vitro exposure to soman and five oximes (toxogonin, HI-6, HLö-7, P2S and 2-PAM) in separate experiments by superfusion. In the absence of soman, toxogonin and HLö-7 in particular induced a concentration dependent significant increase in the evoked release of [(3)H]-acetylcholine. There was also a significant effect of HI-6, but the effect was much smaller. Two pralidoxime salts, P2S (methanesulfonate salt) and 2-PAM (methiodide salt), had similar but lower effects that were only observed at relatively high concentrations. Experiments performed following complete inhibition of the acetylcholinesterase activity by soman (1.0 microM) showed that HI-6 and HLö-7 induced a significant decrease in the potassium-evoked release of [(3)H]-acetylcholine, while the liberation of [(3)H]-choline increased. Toxogonin, P2S and 2-PAM did not reduce significantly the evoked release of [(3)H]-acetylcholine. Only limited reactivation of the acetylcholinesterase activity was observed in superfusion experiments with toxogonin, HI-6, P2S and 2-PAM following exposure of hippocampal slices to soman. However, HLö-7 was proved to be relatively more effective in reactivating the acetylcholinesterase activity at high concentrations (50 and 200 microM). The acetylcholinesterase activity was reactivated to approximately 12% and 40% of control, respectively. It is concluded that HI-6 and HLö-7 have important non-acetylcholinesterase reactivating properties following soman poisoning, as may be seen by the significant reduction in the evoked release of [(3)H]-acetylcholine effected by these oximes. HLö-7 is of particular interest in view of its ability to additionally improve reactivation of the acetylcholinesterase activity.

Acetylcholine↗

PubMed2005

A pilot evaluation of the use of tissue microarrays for quantitation of target distribution in drug discovery pathology.

The use of tissue microarrays (TMAs) in the determination of novel target molecule distribution in organs is an expanding area of discovery pathology. This pilot study was carried out to assess the Chromavision automated cellular imaging system (ACIS) for quantitation of both mRNA and protein distribution in rat and dog TMAs. The targets chosen were a protein kinase, P-CIP2, for mRNA assessment and its downsteam target, peptidylglycine amidating monoxygenase (PAM), for immunohistochemistry (IHC). Oligonucleotide probes produced against P-CIP2, together with an antibody against PAM, were evaluated on rat and dog TMAs. A method for evaluation of target distribution using the ACIS was developed and involved a two-tier approach. Firstly, an initial scanning of the labelled slides identified which tissues expressed the target. Secondly, a more comprehensive analysis was made. This required operator interaction to select specific regions of interest within selected tissue cores and exclude any background labelling from the final assessment. This exacted the level of expression of P-CIP2 or PAM in different cellular populations in tissue cores. A comparative semi-quantitative analysis of the same arrays was concomitantly made by the pathologist in order to assess the relative benefits of a potentially time-consuming detailed morphological evaluation. This involved the histological identification by the pathologist of specific cell populations expressing P-CIP2 or PAM. In this study, we demonstrate the power of an image analysing system to provide quantitative data on target distribution by in situ hybridisation and IHC on normal TMAs. This methodology, together with detailed histological analysis by a pathologist, forms a guideline for future target distribution evaluation within discovery pathology.

Animals↗

PubMed2005

Impact of secondary pulmonary hypertension on lung transplant outcome.

INTRODUCTION: Secondary pulmonary hypertension (SPH), defined as a mean pulmonary artery pressure (PAM) greater than 25 mm Hg, complicates end-stage lung diseases of varying etiology. Although previous studies have suggested that SPH does not adversely affect outcome, no study has assessed the impact of the degree of SPH. METHODS: A retrospective review of the lung transplant database was used to identify patients who underwent either single-lung (SLT) or bilateral lung transplantation (BLT) complicated by SPH. SPH patients were stratified into low SPH (PAM = 30-40 mm Hg) and high SPH (PAM > or = 40 mm Hg). Each group was further sub-categorized into SLT or BLT. Patients with a heart-lung transplant or primary pulmonary hypertension were excluded. Recipients without pulmonary hypertension transplanted over the same time were used as controls. Data are reported as controls vs low SPH vs high SPH. RESULTS: One hundred-four patients received lung transplants between August 1998 and March 2003. There were 45 patients (18 men and 27 women) with SPH. Of these, 28 patients had low SPH, and 17 patients had high SPH. Forty-two patients (18 men and 24 women) without PH were the controls. There were no significant differences between groups except pre-operative oxygen dependence (81% vs 100% vs 94%, respectively) and use of CPB (28.6% vs 57.1% vs 64.7%, respectively). PAO2-PaO2 gradients and PaO2/FIO2 ratios were significantly worse in the high SPH group (116.2 vs 132.9 vs 186.3; p < 0.006) and (277.8 vs 234.3 vs 214.4; p < 0.026) respectively. There was no statistical difference in length of mechanical ventilation or duration of intensive care unit stay between groups. PAMs were significantly different pre-operatively (22.2 +/- 0.8 vs 34.0 +/- 0.6 vs 47.8 +/- 2.0; p < 0.001) and post-operatively (20.9 +/- 1.1 vs 23.7 +/- 1.3 vs 24.8 +/- 2.1; p < 0.001). There were no operative deaths. There were 3 early deaths in the control group, 1 in the low SPH group, and 3 in the high SPH group, none were related to pulmonary hypertension. Actuarial survival at 12, 24, and 48 months was not significantly different among the groups nor between SLT or BLT with SPH. CONCLUSION: Although SPH increases the risk of reperfusion injury; survival is equivalent with mild or moderate pulmonary hypertension. Either SLT or BLT may be used in patients with SPH without compromising outcome. This has the added benefit of expanding the donor pool.

Adult↗

PubMed2005

On the optimality of the genetic code, with the consideration of coevolution theory by comparison of prominent cost measure matrices.

Statistical and biochemical studies have revealed non-random patterns in codon assignments. The canonical genetic code is known to be highly efficient in minimizing the effects of mistranslation errors and point mutations, since it is known that when an amino acid is converted to another due to error, the biochemical properties of the resulted amino acid are usually very similar to those of the original one. In this study, using altered forms of the fitness functions used in the prior studies, we have optimized the parameters involved in the calculation of the error minimizing property of the genetic code so that the genetic code outscores the random codes as much as possible. This work also compares two prominent matrices, the Mutation Matrix and Point Accepted Mutations 74-100 (PAM(74-100)). It has been resulted that the hypothetical properties of the coevolution theory of the genetic code are already considered in PAM(74-100), giving more evidence on the existence of bias towards the genetic code in this matrix. Furthermore, our results indicate that PAM(74-100) is biased towards the single base mistranslation occurrences in second codon position as well as the frequency of amino acids. Thus PAM(74-100) is not a suitable substitution matrix for the studies conducted on the evolution of the genetic code.

Animals↗

PubMed2006

Stimulation of mast cells via FcvarepsilonR1 and TLR2: the type of ligand determines the outcome.

Little is known about the interplay between pathophysiological processes of allergy and infection, particularly with respect to mast cell (MC)-mediated responses. The presence and recognition of pathogen-associated molecular patterns (PAMPs) might have broad impact on the development and severity of diseases. In this study, we assessed the influence of toll-like receptor 2 (TLR 2)-dependent synthetic analogs of bacterial lipopeptides (LPs), Pam(3)CSK(4) and MALP-2, on Ag (DNP-HSA)-triggered responses in bone marrow-derived MCs (BMMCs). Both LPs strongly synergized with sub-optimal amounts of Ag in the stimulation of cytokine release. Intriguingly, Pam(3)CSK(4), but not MALP-2 suppressed Ag-induced degranulation of BMMCs (together with early tyrosine phosphorylation and calcium mobilization) in a TLR2-independent manner. Further analysis revealed that Pam(3)CSK(4), most probably by electrostatic forces, reduced the level of active DNP-HSA and that this, in turn, was responsible for the suppression of Ag-induced degranulation. Thus, our work demonstrates that LPs can synergize with IgE+Ag in stimulating the production of IL-6 by BMMCs. As well, our findings with Pam(3)CSK(4) indicate that one must be cautious when interpretating results obtained with "model" substances and the combination of ligands must be carefully chosen when functional interactions between the high-affinity receptor for IgE (FcepsilonR1) and TLR2 are examined.

Animals↗

PubMed2006

Human parotid saliva contains soluble toll-like receptor (TLR) 2 and modulates TLR2-mediated interleukin-8 production by monocytic cells.

Toll-like receptor (TLR) family members are pattern-recognition receptors and very important molecules in innate immunity. Although TLRs are originally type I transmembrane receptors, soluble forms of TLRs are detected in human plasma and milk. This study showed that soluble TLR2 (sTLR2) is detected in human parotid saliva. Western blotting with anti-TLR2 antibodies (Abs) showed that three polypeptides are detected as sTLR2 with molecular weights of 55, 40 and 27kDa, respectively. Parotid saliva neutralized the binding of anti-TLR2 polyclonal Ab to cell-surface TLR2 on THP-1, a human monocytic cell line. Immunohistochemical analysis revealed that TLR2 is expressed in serous and interlobular ductal cells of human salivary gland. Human salivary gland cell lines, AZA3 and HSY, constitutively expressed TLR2. Parotid saliva augmented IL-8 production of THP-1 cells stimulated with a synthetic TLR2 ligand, Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)). Depletion of sCD14 from parotid saliva by immunoprecipitation eliminated the augmentation of IL-8 production, indicating that the augmentable effects depended on sCD14 in parotid saliva. On the other hand, preincubation of Pam(3)CSK(4) with parotid saliva abrogated the augmentation of IL-8 production, indicating that sTLR2 in saliva bound to Pam(3)CSK(4) and neutralized its function. These results suggest that parotid saliva modulates the TLR2-mediated immune responses with binary mechanisms via sTLR2 and sCD14 in the oral cavity.

Cell Line, Tumor↗

PubMed2004

Methenamine silver staining quantitative digital histochemistry in chronic allograft nephropathy.

Renal function and final outcome of renal allografts have been correlated with irreversible damage. This study describes a quantitative histochemical method relying on periodic acid methenamine silver (PAMS) staining of all renal compartments. Among 60 renal allograft biopsies from 43 patients, 15 biopsies showing pure chronic allograft nephropathy were selected to determine PAMS-stained area percentage (SAP), using image analysis with quantitative histochemistry. Of the 15 cases, 9 (60%) were grade I and 6 (40%) were grade II chronic allograft nephropathy (CAN). The mean serum creatinine (sCr) value was 1.86 +/- 0.47 for allograft biopsies. The mean (+/-SD) SAP for the implantation biopsies was 10.58 +/- 1.87%, and for allograft biopsies 25.26 +/- 9.67 (P <<.000). The serum creatinine (sCr) values for grade I versus II CAN were 1.63 +/- 0.24 versus 2.20 +/- 0.54 mg/dL, respectively (P=.019), and SAP values were 18.97 +/- 0.24 versus 34.7 +/- 5.89 (P=.003). There was a strong positive correlation between sCr values and SAP (P=.005; r=0.64). These findings show the PAMS approach to be a useful alternative method for reflecting damage in more than one compartment of the renal tissue. Also, the method can discriminated implantation and allograft biopsies as well as grade I and II CAN cases. The series is small for a multivariate analysis of the value of SAP measurements in PAMS-stained sections as a prognosticator, but the data support its use.

Biopsy↗

PubMed2004

Applications of linear-motor-driven photoacoustic microscope.

A photoacoustic microscope (PAM) with linear-motor-drives was designed and fabricated. The PAM has the performance of high spatial resolution, fast scan speed, and quietness at scanning compared used a PAM with conventional pulse-motor slide stages. The PAM was applied to image semiconductor devices and subsurface defects and to measure the amount of the pollen of Cryptomeria japonica (Taxodiaceae). In the former case, the spatial resolution was about 20 microm. In the latter case, PA amplitude image had a good resolution to be resolved up to few single pollen particles. In addition, the obtained PA amplitude image was integrated over the specimen surface, and the integrated signal was measured at various pollen mass. The calibration curve showed the correlation value of 0.94.

Acoustics↗

PubMed2005

Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection.

Porcine respiratory and reproductive syndrome virus (PRRSV) disease, one of the most economically significant viral diseases in the swine industry, is characterized by miscarriages, premature farrowing, stillborn pigs, and respiratory disease associated with death and chronic poor performance of nursing and weaned pigs. Interleukin-12 (IL-12) is a key component in driving the development of cell-mediated immunity as well as stimulating interferon-gamma (IFN-gamma) production from T cells and natural killer cells. Although some studies have investigated the use of IL-12 as a vaccine adjuvant in swine, little is known about its effectiveness as a treatment against viral diseases in swine. The present study investigated whether recombinant porcine IL-12 (rpIL-12) enhances the immune response and thereby diminishes the effects of PRRSV infection in young pigs. Interestingly, in vitro experiments demonstrated that rpIL-12 is capable of inducing swine pulmonary alveolar macrophages (PAMs), the target cells of PRRSV, to produce IFN-gamma in a dose and time dependent manner. In addition, in vitro studies also revealed that rpIL-12 treatment was capable of significantly reducing PRRSV viral titers in PAMs. In vivo administration of rpIL-12 significantly decreased PRRSV titers in the lungs and blood of infected animals. Furthermore, treatment with rpIL-12 prevented significant growth retardation in PRRSV-infected animals. Finally, in response to viral antigen recall challenge, PAMs isolated from rpIL-12-treated/PRRSV-infected animals produced greater amounts of IFN-gamma and lesser amounts of interleukin-10 than PAMs isolated from non-rpIL-12-treated/PRRSV-infected animals. Taken together our data indicate that treatment with rpIL-12 may provide an effective approach to control or ameliorate PRRSV-induced disease in swine.

Animals↗

PubMed2025

RNA-DNA hybrid binding domain broadens the editing window of base editors.

Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.

Gene Editing↗

PubMed2003

Glutathione, S-substituted glutathiones, and leukotriene C4 as substrates for peptidylglycine alpha-amidating monooxygenase.

The C-terminal alpha-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine alpha-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C(4) results in the consumption of O(2) and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal alpha-hydroxyglycine-extended peptide. Amidated glutathione (gammaGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest (V/K)(app) values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V/K)(app) of the substrates described in this report.

Amino Acids↗

PubMed1985

gamma-Interferon stimulates production of 1,25-dihydroxyvitamin D3 by normal human macrophages.

We show for the first time that normal human pulmonary alveolar macrophages (PAM) markedly enhance their basal rate of the production of [3H]-1,25(OH)2D3 when cultured in the presence of recombinant gamma-interferon (gamma-IFN). The rate of conversion of [3H]-25(OH)D3 to [3H]-1,25(OH)2D3 was dose-dependent in a linear fashion. A maximal production of 1,25(OH)2D3 by PAM occurred after exposure of PAM to gamma-IFN for one day. This maximum plateau-level was sustained for at least five days. The authenticity of the putative 1,25(OH)2D3 obtained from PAM was tested by demonstrating the exact comigration of [3H]-1,25(OH)2D3 with chemically synthesized 1,25(OH)2D3 in four different HPLC-systems.

Animals↗

PubMed1987

Reduced thiol content in L1210 cells treated with BSO increases DNA crosslinking by melphalan.

Endogenous thiols such as glutathione (GSH) are known to mediate the activity of bifunctional alkylating agents such as melphalan (L-PAM). GSH levels can be reduced by cell growth in the presence of an analog of gamma glutamyl, alpha-aminobutyrate, L-buthionine (S/R) sulfoximine (L-BSO). L-1210 murine lymphocytic leukemia cells grown in vitro in the presence of 10 microM less than 15% L-BSO possessed 1% of the normal GSH levels. This pretreatment regimen did not significantly alter cell viability but did enhance the cytotoxicity produced by a 15 microM-1hr L-PAM treatment. The increased cell killing correlated with enhanced DNA-DNA crosslinking immediately following L-PAM exposure. No effect of BSO pretreatments on the incomplete removal of crosslinks over 36 hr of observation was seen. These results suggest that GSH levels modulate the initial degree of L-PAM-induced DNA crosslinking, but not the long term repair of these lesions.

Animals↗

PubMed1989

Ontogenetic expression of peptidyl-glycine alpha-amidating monooxygenase mRNA in the rat pancreas.

Qualitative and quantitative expression of m.RNA coding for Peptidyl-Glycine alpha-Amidating Monooxygenase (PAM) in the developing rat pancreas was investigated by Northern and dot blot hybridization, with a bovine PAM c.DNA probe (0.7 kb fragment). A specific hybridization signal was evidenced for a 3.7 kb m.RNA species. Measurement of PAM m. RNA rate during the rat pancreas ontogenesis revealed a biphasic profile which appeared corelated with that of gastrin and TRH m.RNA respectively. On the other hand, streptozotocin-treatment resulted in a 50% decrease of PAM m.RNA levels.

Aging↗

PubMed2002

Affinity purification of polyclonal antibodies using a new all-D synthetic peptide ligand: comparison with protein A and protein G.

This study investigates the applicability of the protein A Mimetic (PAM) affinity ligand, obtained from the screening of a multimeric combinatorial peptide library, in immunoglobulin isolation from serum. To avoid protease degradation, the ligand has been substituted by its inverso form, named D-PAM, synthesized by replacing all amino acids with the corresponding D derivatives. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture IgG directly from the serum in a single chromatographic step, with a recovery yield ranging from 60% to 90%, a purity degree higher than 90%, and with a full recovery of antibody activity. Column capacity, determined by applying a large excess of purified IgG to 1 ml bed volume column, was close to 52 mg/ml for bovine IgG, 58 mg/ml for goat IgG, 66 mg/ml for horse IgG, 50 mg/ml for human IgG, 52 mg/ml for mouse IgG, 36 mg/ml for rabbit IgG and 48 mg/ml for sheep IgG. D-PAM peptide was found to be very stable to protease activity, and after prolonged incubation with mouse serum. Similarly, the corresponding derivatized matrix tested before and after various treatments, including sanitization and autoclaving procedures maintained its IgG binding properties, thus indicating a very high stability in terms of ligand leakage and degradation.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria.

BACKGROUND: Pregnancy-associated malaria caused by Plasmodium falciparum adherence to chondroitin sulfate A in the placental intervillous space is a major cause of low birthweight and maternal anaemia in areas of endemic P falciparum transmission. Adhesion-blocking antibodies that specifically recognise parasite-encoded variant surface antigens (VSA) are associated with resistance to pregnancy-associated malaria. We looked for a possible relation between VSA-specific antibody concentrations, placental infection, and protection from low birthweight and maternal anaemia. METHODS: We used flow cytometry to measure VSA-specific IgG concentrations in plasma samples taken during child birth from 477 Kenyan women selected from a cohort of 910 women on the basis of HIV-1 status, gravidity, and placental histology. We measured VSA expressed by one placental P falciparum isolate and two isolates selected or not selected for chondroitin sulfate A adhesiveness in-vitro. FINDINGS: Concentrations of plasma IgG specific for VSA, expressed by chondroitin sulfate A-adhering parasites (VSA in pregnancy-associated malaria or vsa-pam), increased with gravidity and were associated with placental histological findings. Women with chronic pregnancy-associated malaria and low or absent VSA-PAM-specific IgG had lower haemoglobin values (reduced by 17 g/L; 95% CI 8.1-25.2) and delivered smaller babies (birthweight reduced by 0.26 kg; 0.10-0.55) than did corresponding women with high VSA-PAM-specific IgG. No such relation was shown for concentrations of IgG with specificity for non-pregnancy-associated malaria VSA. INTERPRETATION: VSA-PAM-specific IgG protects against low birthweight and maternal anaemia. Our data indicate an important mechanism of clinical protection against malaria and raise hope for the clinical effectiveness of a potential VSA-based vaccine against pregnancy-associated malaria.

Animals↗

PubMed2000

Effect of a neutral citrate solution on the fluoride release of resin-modified glass ionomer and polyacid-modified composite resin cements.

The effect of 0.01 mol/l citrate solution at pH = 7 on the fluoride release is compared for the resin-modified glass ionomer cements (RM-GIC) GC Lining LC, PhotacBond, Vitremer and Vitrebond and for the polyacid-modified composite resins (PAM-C) Variglass and Dyract by means of the six-month fluoride release profiles at 37 degrees C. The fluoride release of both RM-GIC and PAM-C increases in the neutral citrate solution as compared to water, which can be explained by the ability of citrate to complex metal ions and hence to degrade the glass as well as the polysalt matrix of the cement. Although RM-GIC release more fluoride than PAM-C in water as well as in citrate solution, the relative increase in fluoride release upon immersion in citrate solution is most pronounced for PAM-C. Whereas for the latter citrate affects both the short-term and long-term fluoride release, for RM-GIC only the long-term fluoride release is affected. This suggests that the action of citrate increases with decreasing importance of the polysalt formation in the hardening of the material. This could be explained on the basis of the difference in the chemical properties of the cement matrix.

Acrylic Resins↗

PubMed1986

A comparison of visual function tests in eyes with maculopathy.

Several recently developed tests of visual function, including the Potential Acuity Meter (PAM), laser interferometer (LI), white-light interferometer (WLI), blue field entoptic phenomenon, and focal electroretinogram (ERG) were compared in 81 eyes with clear media and known macular disease. The results indicate that the PAM, the LI, and the WLI overread relative to Snellen acuity. Laser interferometric acuity values differed from Snellen acuity by at least 1.5 octaves in approximately 40% of all eyes, regardless of stimulus size (2, 5, or 8 degrees). Similar results were obtained with the WLI. Agreement with Snellen acuity was better for the PAM, with 91% of eyes falling within 1.5 octaves of Snellen acuity. Blue field and focal ERG results were categorized as normal or abnormal. While not producing Snellen equivalents, abnormal results from the blue field and focal ERG corresponded with poor Snellen acuity (less than 20/40) in 65% and 91% of eyes, respectively. Assuming that media opacities do not prevent adequate retinal stimulation, the present results suggest that the PAM and focal ERG are the most reliable for evaluating macular function when maculopathy is present.

Electroretinography↗