Relationship between antifungal activity and hydrophobicity of kojic acid derivatives

Twenty three kojic acid derivatives were tested in the agar diffusion test against twenty five dermatophytic fungi. The inhibitory effects at equal doses (10 mg/L) were correlated with the 1-octanol — water partition coefficients of the compounds. An improvement in antifungal activity can be achieved by increasing the hydrophobicity of the compounds. This work was supported by Slovak Grant Agency (grant no. 1/4404b4/42).     

Caspase-14 but not caspase-3 is processed during the development of fetal mouse epidermis

The activation of caspases is a central step in apoptosis and may also be critical for terminal differentiation of epidermal keratinocytes (KC). In particular, caspase-3 has been implicated in the differentiation of embryonic KC as well as in programmed cell death of KC, and caspase-14 has been suggested to function in the formation or homeostasis of the stratum corneum (SC). To test the putative roles of these proteases, we determined their expression level and activation status during development of fetal mouse epidermis. The level of procaspase-3 did not change significantly during epidermal development, and enzyme activation was undetectable at any timepoint investigated. Despite the lack of active caspase-3, the newly formed stratum granulosum and the regressing periderm contained cells positive in the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, indicating that nuclear DNA was degraded without activation of caspase-3, thereby arguing against a proteolytic function of caspase-3 in embryonic KC differentiation. By contrast, caspase-14 increased in abundance from embryonic day 14.5 (E14.5) onwards and consistently localized to the suprabasal layers of fetal epidermis. The caspase-14 pro-enzyme was processed into its catalytic subunits, a step required for enzyme activity, on day E17.5, coinciding with SC formation. Thus, processing of procaspase-14 is not confined to air-exposed mature skin but also occurs during epidermal development in utero. In summary, this study demonstrates that caspase-14, but not caspase-3 activation coincides temporally and spatially with embryonic KC differentiation, suggesting a role for caspase-14 in terminally differentiated KC.     

The elusive indigo precursors in woad (Isatis tinctoria L.)--identification of the major indigo precursor, isatan A, and a structure revision of isatan B

A metabolite-profiling study of shock-frozen leaves of Isatis tinctoria L., an old indigo dye plant and medicinal herb, revealed a complex pattern of indigo-forming compounds with higher polarities than the known indigo precursors isatan B and indican. These highly unstable compounds underwent rapid post-harvest transformation and were not detected in air-dried leaves. The major indigo precursor, named isatan A (4), was isolated by rapid normal-phase and gel chromatography, along with isatan B (3). A full spectral data set of 3 showed that the previous structure assignment as 'indoxyl-5-ketogluconate' has to be revised to 1H-indol-3-yl beta-D-ribohex-3-ulopyranoside. Isatan A (4) was identified as 1H-indol-3-yl 6'-O-(carboxyacetyl)-beta-D-ribohex-3'-ulopyranoside. In aqueous solution, glycosides 3 and 4 occur as hydrates and undergo rapid hydrolysis under very mild acidic or basic conditions.     

PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.     

Effectiveness of Home-Based Cupping Massage Compared to Progressive Muscle Relaxation in Patients with Chronic Neck Pain—A Randomized Controlled Trial

Chronic neck pain is a major public health problem with very few evidence-based complementary treatment options. This study aimed to test the efficacy of 12 weeks of a partner-delivered home-based cupping massage, compared to the same period of progressive muscle relaxation in patients with chronic non-specific neck pain. Patients were randomly assigned to self-directed cupping massage or progressive muscle relaxation. They were trained and asked to undertake the assigned treatment twice weekly for 12 weeks. Primary outcome measure was the current neck pain intensity (0–100 mm visual analog scale; VAS) after 12 weeks. Secondary outcome measures included pain on motion, affective pain perception, functional disability, psychological distress, wellbeing, health-related quality of life, pressure pain thresholds and adverse events. Sixty one patients (54.1±12.7 years; 73.8%female) were randomized to cupping massage (n = 30) or progressive muscle relaxation (n = 31). After treatment, both groups showed significantly less pain compared to baseline however without significant group differences. Significant effects in favor of cupping massage were only found for wellbeing and pressure pain thresholds. In conclusion, cupping massage is no more effective than progressive muscle relaxation in reducing chronic non-specific neck pain. Both therapies can be easily used at home and can reduce pain to a minimal clinically relevant extent. Cupping massage may however be better than PMR in improving well-being and decreasing pressure pain sensitivity but more studies with larger samples and longer follow-up periods are needed to confirm these results.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01500330","term_id":"NCT01500330"}}NCT01500330     

A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells

BACKGROUND: Early clinical trials for gene therapy of human gliomas with retroviral packaging cells (PC) have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor. In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus (LCMV) based on tumor-infiltrating progenitor cells. METHODS: Tumor-infiltrating progenitor cells, which had been isolated from adult rat bone marrow (BM-TIC), were modified to stably express Gag-Pol proteins of moloney murine leukemia virus (Mo-MLV) and glycoproteins of LCMV. Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines. Additionally, gene transfer was tested in a rat glioma model in vivo. RESULTS: The BM-TIC-derived packaging cell line (BM-TIPC) produced retroviral vectors with titers between 2-8 x 10(3) transducing units (TU)/ml. Extended culturing of BM-TIPC over several weeks and freezing/thawing of cells did not affect vector titers. No replication-competent retrovirus was released from BM-TIPC. In a rat glioma model, BM-TIPC infiltrated the tumors extensively and with high specificity. Moreover, BM-TIPC mediated transduction of glioma cells in vivo. CONCLUSION: This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors. These BM-TIPC may be a useful tool to enhance specificity and efficacy of gene transfer to gliomas in patients.     

Immune Suppressive Effect of Cinnamaldehyde Due to Inhibition of Proliferation and Induction of Apoptosis in Immune Cells: Implications in Cancer

Background

Besides its anti-inflammatory effects, cinnamaldehyde has been reported to have anti-carcinogenic activity. Here, we investigated its impact on immune cells.

Methods

Activation of nuclear factor-κB by cinnamaldehyde (0–10 µg/ml) alone or in combination with lipopolysaccharide was assessed in THP1XBlue human monocytic cell line and in human peripheral blood mononuclear cells (PBMCs). Proliferation and secretion of cytokines (IL10 and TNFα) was determined in primary immune cells and the human cell lines (THP1, Jurkat E6-1 and Raji cell lines) stimulated with cinnamaldehyde alone or in conjunction with lipopolysaccharide. Nitric oxide was determined in mouse RAW264.7 cells. Moreover, different treated PBMCs were stained for CD3, CD20 and AnnexinV.

Results

Low concentrations (up to 1 µg/ml) of cinnamaldehyde resulted in a slight increase in nuclar factor-kB activation, whereas higher concentrations led to a dose-dependent decrease of nuclear factor-kB activation (up to 50%) in lipopolysachharide-stimulated THP1 cells and PBMCs. Accordingly, nitric oxide, interleukin 10 secretion as well as cell proliferation were reduced in lipopolysachharide-stimulated RAW264.7 cells, PBMCs and THP1, Raji and Jurkat-E6 immune cells in the presence of cinnamaldehyde in a concentration-dependent manner. Flow cytometric analysis of PBMCs revealed that CD3+ were more affected than CD20+ cells to apopotosis by cinnamaldehyde.

Conclusion

We attribute the anti-inflammatory properties of cinnamaldehyde to its ability to block nuclear factor-κB activation in immune cells. Treatment with cinnamaldehyde led to inhibition of cell viability, proliferation and induced apoptosis in a dose-dependent manner in primary and immortalized immune cells. Therefore, despite its described anti-carcinogenic property, treatment with cinnamaldehyde in cancer patients might be contraindicated due to its ability to inhibit immune cell activation.     

Autophagy in the thymic epithelium is dispensable for the development of self-tolerance in a novel mouse model

The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14), including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f) showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP)-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre). Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1) in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+) CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.