K+ channels and the microglial respiratory burst

Microglial activation following central nervous system damage or disease often culminates in a respiratory burst that is necessary for antimicrobial function, but, paradoxically, can damage bystander cells. We show that several K+ channels are expressed and play a role in the respiratory burst of cultured rat microglia. Three pharmacologically separable K+ currents had properties of Kv1.3 and the Ca2+/calmodulin-gated channels, SK2, SK3, and SK4. mRNA was detected for Kv1.3, Kv1.5, SK2, and/or SK3, and SK4. Protein was detected for Kv1.3, Kv1.5, and SK3 (selective SK2 and SK4 antibodies not available). No Kv1.5-like current was detected, and confocal immunofluorescence showed the protein to be subcellular, in contrast to the robust membrane localization of Kv1.3. To determine whether any of these channels play a role in microglial activation, a respiratory burst was stimulated with phorbol 12-myristate 13-acetate and measured using a single cell, fluorescence-based dihydrorhodamine 123 assay. The respiratory burst was markedly inhibited by blockers of SK2 (apamin) and SK4 channels (clotrimazole and charybdotoxin), and to a lesser extent, by the potent Kv1.3 blocker agitoxin-2.     

Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.     

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1)

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.     

The importance of homologous recombination in the generation of large deletions in hybrid plasmids in Amycolatopsis mediterranei

The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined. pT3.2I is 15,390 bp long with a 53.7% GC content. Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp). Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids. Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB. Based on sequence comparisons, pT3. 2I can be related to plasmids in the pColE2-CA42 incB incompatibility group.     

Antiinflammatory activity of Muktashukti bhasma.

Muktashukti bhasma (MSB), an Ayurvedic compound, consisting of pearl, Aloe vera and vinegar, inhibited acute and subacute inflammation in albino rats as induced by subplanter injection of carrageenan, histamine, 5-HT, nystatin and subcutaneous implant of cotton pellets. In all the test procedures the antiinflammatory response of 1000 mg/kg MSB was comparable to the response observed with 300 mg/kg acetylsalicylic acid (ASA). Oral premedication with MSB delayed castor oil-induced diarrhoea in rats, indicating its prostaglandin inhibitory activity. The antiinflammatory activity of the compound is attributed to its ability to cause inhibition of prostaglandins, histamine and 5-HT and also by stabilization of the lysosomal membranes. The antiinflammatory activity of MSB seems one third to half as potent as ASA.     

Silver-Zinc Redox-Coupled Electroceutical Wound Dressing Disrupts Bacterial Biofilm

Pseudomonas aeruginosa biofilm is commonly associated with chronic wound infection. A FDA approved wireless electroceutical dressing (WED), which in the presence of conductive wound exudate gets activated to generate electric field (0.3–0.9V), was investigated for its anti-biofilm properties. Growth of pathogenic P. aeruginosa strain PAO1 in LB media was markedly arrested in the presence of the WED. Scanning electron microscopy demonstrated that WED markedly disrupted biofilm integrity in a setting where silver dressing was ineffective. Biofilm thickness and number of live bacterial cells were decreased in the presence of WED. Quorum sensing genes lasR and rhlR and activity of electric field sensitive enzyme, glycerol-3-phosphate dehydrogenase was also repressed by WED. This work provides first electron paramagnetic resonance spectroscopy evidence demonstrating that WED serves as a spontaneous source of reactive oxygen species. Redox-sensitive multidrug efflux systems mexAB and mexEF were repressed by WED. Taken together, these observations provide first evidence supporting the anti-biofilm properties of WED.     

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase

Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01196871","term_id":"NCT01196871"}}NCT01196871