Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.     

A New In Vitro Model to Study Cellular Responses after Thermomechanical Damage in Monolayer Cultures

Although electrosurgical instruments are widely used in surgery to cut tissue layers or to achieve hemostasis by coagulation (electrocautery), only little information is available concerning the inflammatory or immune response towards the debris generated. Given the elevated local temperatures required for successful electrocautery, the remaining debris is likely to contain a plethora of compounds entirely novel to the intracorporal setting. A very common in vitro method to study cell migration after mechanical damage is the scratch assay, however, there is no established model for thermomechanical damage to characterise cellular reactions. In this study, we established a new in vitro model to investigate exposure to high temperature in a carefully controlled cell culture system. Heatable thermostat-controlled aluminium stamps were developed to induce local damage in primary human umbilical vein endothelial cells (HUVEC). The thermomechanical damage invoked is reproducibly locally confined, therefore allowing studies, under the same experimental conditions, of cells affected to various degrees as well as of unaffected cells. We show that the unaffected cells surrounding the thermomechanical damage zone are able to migrate into the damaged area, resulting in a complete closure of the ‘wound’ within 48 h. Initial studies have shown that there are significant morphological and biological differences in endothelial cells after thermomechanical damage compared to the mechanical damage inflicted by using the unheated stamp as a control. Accordingly, after thermomechanical damage, cell death as well as cell protection programs were activated. Mononuclear cells adhered in the area adjacent to thermomechanical damage, but not to the zone of mechanical damage. Therefore, our model can help to understand the differences in wound healing during the early phase of regeneration after thermomechanical vs. mechanical damage. Furthermore, this model lends itself to study the response of other cells, thus broadening the range of thermal injuries that can be analysed.     

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon × Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0–34.4 and 28.9–31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14–70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.     

Hepatic zinc response via metallothionein induction after tumor transplantation

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc-MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When (65)ZnCl(2) was intravenously injected, liver (65)Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood (65)Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, (65)Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. (65)Zn uptake in the tumor and blood (65)Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc-MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.     

Blood oxygenation using microbubble suspensions

Microbubbles have been used in a variety of fields and have unique properties, for example shrinking collapse, long lifetime, efficient gas solubility, a negatively charged surface, and the ability to produce free radicals. In medicine, microbubbles have been used mainly as diagnostic aids to scan various organs of the body, and they have recently been investigated for use in drug and gene delivery. However, there have been no reports of blood oxygenation by use of oxygen microbubble fluids without shell reagents. In this study, we demonstrated that nano or microbubbles can achieve oxygen supersaturation of fluids, and may be sufficiently small and safe for infusion into blood vessels. Although Po(2) increases in fluids resulting from use of microbubbles were inhibited by polar solvents, normal saline solution (NSS) was little affected. Thus, NSS is suitable for production of oxygen-rich fluid. In addition, oxygen microbubble NSS effectively improved hypoxic conditions in blood. Thus, use of oxygen microbubble (nanobubble) fluids is a potentially effective novel method for oxygenation of hypoxic tissues, for infection control, and for anticancer treatment.     

Fibromyalgia syndrome and chronotype: late chronotypes are more affected

Sleep has strong links to the symptomology of fibromyalgia syndrome (FMS), a diffuse musculoskeletal pain disorder. Information about the involvement of the circadian clock is, however, sparse. In this study, 1548 individuals with FMS completed an online survey containing questions on demographics, stimulant consumption, sleep quality, well-being and subjective pain, chronotype (assessed by the Munich ChronoType Questionnaire, MCTQ), and FMS impact. Chronotype (expressed as the mid-sleep-point on free days, corrected for sleep deficit on workdays, MSF(sc)) significantly correlated with stress-ratings, so-called "memory failures in everyday life," fatigue, FMS impact, and depression but not with anxiety. When chronotypes were categorized into 3 groups (early, intermediate, late), significant group differences were found for sum scores of perceived stress, memory failures in everyday life, fatigue, FMS impact, and depression but not anxiety, with late chronotypes being more affected than early chronotypes. Sleepiness ratings were highest in early chronotypes. Challenges of sleep quality and subjective pain were significantly increased in both early and late chronotypes. The results show that according to their reports, late chronotypes are more affected by fibromyalgia.     

Association of IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency

Background

The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely to be influenced by modifier genes. Genome-wide association studies and integrative genomics approaches in COPD have demonstrated significant associations with SNPs in the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2).We investigated whether SNPs in the chromosome 15q region would be modifiers for lung function and COPD in AAT deficiency.

Methods

The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry. Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEV₁ percent of predicted and FEV₁/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed in the AAT Genetic Modifiers Study. In the replication set, general linear models were used for quantitative phenotypes and logistic regression models were used for the presence/absence of emphysema or COPD.

Results

Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV₁ percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730) were associated with the post-bronchodilator FEV₁ percent of predicted and pre-bronchodilator FEV₁/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry dataset, rs2568494 was significantly associated with emphysema in the male subgroup; significant SNP-by-smoking interactions were observed.

Conclusions

IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT deficiency and may be sex-specific in their impact.     

Biomarkers of Good EULAR Response to the B Cell Depletion Therapy in All Seropositive Rheumatoid Arthritis Patients: Clues for the Pathogenesis

Objective

To find out whether a high number of auto-antibodies can increase the probability of a “good-EULAR response” and to identify the possible biomarkers of response in seropositive rheumatoid arthritis (RA) patients undergoing the B cell depletion therapy (BCDT).

Patients and Methods

One hundred and thirty-eight patients with long standing RA (LSRA), 75% non or poorly responsive to one or more TNFα blockers, all seropositive for at least one autoantibody (AAB) (RF-IgM, RF-IgA, RF-IgG, anti-MCV, ACPA-IgG, ACPA-IgA, ACPA-IgM) received one full course of BCDT. The major outcomes (moderate or good-EULAR response) were assessed after 6 months of therapy. The IL6 and BAFF levels were also determined.

Results

At a 6-month follow-up, 33 (23.9%) of the RA patients achieved a good EULAR response. Having up to 5-AABs positivity increased the chances for treatment response. After a logistic regression analysis, however, only 4 baseline factors arose as associated with a good-EULAR response: no steroid therapy (OR = 6.25), a lymphocyte count <1875/uL (OR = 10.74), a RF-IgG level >52.1 IU/ml (OR = 8.37) and BAFF levels <1011 pg/ml (OR = 7.38). When all the AABs, except for RF-IgM and ACPA-IgG, were left in the analysis, the two final predictors were no-steroid therapy and low lymphocyte count.

Discussion

The number of AABs increased the chances of being a “good-EULAR” responder. The only predictors, however, at the baseline of a good response in this seropositive cohort of RA patients were 2 simple variables – no steroids and lymphocyte count – and two laboratory assays – IgG-RF and BAFF.     

Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.