Kristina Todorova

The kinase p38|[alpha]| serves cell type|[ndash]|specific inflammatory functions in skin injury and coordinates pro- and anti-inflammatory gene expression

The mitogen-activated protein kinase p38 mediates cellular responses to injurious stress and immune signaling. Among the many p38 isoforms, p38α is the most widely expressed in adult tissues and can be targeted by various pharmacological inhibitors. Here we investigated how p38α activation is linked to cell type–specific outputs in mouse models of cutaneous inflammation. We found that both myeloid and epithelial p38α elicit inflammatory responses, yet p38α signaling in each cell type served distinct inflammatory functions and varied depending on the mode of skin irritation. In addition, myeloid p38α limited acute inflammation via activation of anti-inflammatory gene expression dependent on mitogen- and stress-activated kinases. Our results suggest a dual function for p38α in the regulation of inflammation and show mixed potential for its inhibition as a therapeutic strategy.

Phosphatase activity and copper uptake during growth of Aspergillus niger

Abstract The fungus Aspergillus niger produced extracellular and cellular acid phosphatase activities during growth in the presence or absences of copper ions in the medium. The levels of both phosphatase activities depended on the copper concentrations in the medium. Enzyme activities were maximal at the mid-exponential growth phase, as well as at the higher copper concentration and decreased as growth time increased from 12 to 48 h. The total uptake of copper (II) by mycelia, growing at the presence of copper was highest when the levels of enzyme activities were maximal. Between 10 and 20% copper (II) was not removed by washing in 0.1 M H2SO4, suggesting that this copper (II) was bound intracellularly by mycelia of different age. On the other hand, copper ions slightly inhibited cellular acid phosphatase activity of Aspergillus niger with a Ki of 0.89 mM. Acid phosphatase overproduction, as well as copper uptake of mycelia indicated a possible participation of acid phosphatases in the resistance of the fungal strain to the copper toxicity.

Small GTPase RhoE/Rnd3 is a critical regulator of Notch1 signaling.

Aberrations of Notch signaling have been implicated in a variety of human cancers. Oncogenic mutations in NOTCH1 are common in human T-cell leukemia and lymphomas. However, loss-of-function somatic mutations in NOTCH1 arising in solid tumors imply a tumor suppressor function, which highlights the need to understand Notch signaling more completely. Here, we describe the small GTPase RhoE/Rnd3 as a downstream mediator of Notch signaling in squamous cell carcinomas (SCC) that arise in skin epithelia. RhoE is a transcriptional target of activated Notch1, which is attenuated broadly in SCC cells. RhoE depletion suppresses Notch1-mediated signaling in vitro, rendering primary keratinocytes resistant to Notch1-mediated differentiation and thereby favoring a proliferative cell fate. Mechanistic investigations indicated that RhoE controls a key step in Notch1 signaling by mediating nuclear translocation of the activated portion of Notch1 (N1IC) through interaction with importins. Our results define RhoE as a Notch1 target that is essential for recruitment of N1IC to the promoters of Notch1 target genes, establishing a regulatory feedback loop in Notch1 signaling. This molecular circuitry may inform distinct cell fate decisions to Notch1 in epithelial tissues, where carcinomas such as SCC arise.

Fluorescence Studies on Denaturation and Stability of Recombinant Human Interferon-Gamma

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNγ) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 m and ΔG0 = 13.4 kJ/mol) than urea (C* = 2.8 m and ΔG0 = 11.7 kJ/mol). The close ΔG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNγ is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNγ remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNγ, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNγ.

Role of the C-terminal chain in human interferonγ stability: An electrostatic study

Electrostatic interactions in two structures of human interferon gamma (hIFNγ), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model. It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH. The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNγ molecule rather than an artifact of the crystal packing. His111 was also found deprotonated at neutral pH. It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions. The structure corresponding to hIFNγ in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites. This finding suggested that on receptor binding, hIFNγ adopts energetically more favorable, relaxed, conformation. It was experimentally shown that in contrast to the full‐size hIFNγ, the construct having 21 amino acid residues deleted from the C‐terminus is soluble. The hydrophobicity profile analysis suggested that factors other than the exposure of hydrophobic parts of the molecule are responsible for the low stability and propensity for aggregation. On the basis of these results, it was assumed that the electrostatic influence of the C‐terminal part contributes particularly to the low solvent exposure of the titratable groups, and hence to the low structural stability and propensity for aggregation of the recombinant hIFNγ. Proteins 2001;43:125–133.

Influence of interferons on the repair of UV-damaged DNA.

The capacity for nucleotide excision repair of a normal (WISH) and three tumour (MCF-7, HeLa, Namalva) cell lines treated with human recombinant interferons (hrIFN-α and hrIFN-γ) was compared by the host cell reactivation assay. The cells were transfected with in vitro UV-damaged plasmid DNA (pEGFP-N1). The repair capacity was determined by measuring the fluorescence intensity of the expressed marker protein in total cell lysates. The correlation between the interferon-induced NO content and the suppressive effect of interferons on DNA repair was shown. The decrease of repair activity and NO induction by hrIFN-α were greatest in WISH, followed by MCF-7, Namalva and HeLa cells, whereas hrIFN-γ was the best NO inducer and inhibitor for the repair of Namalva, followed by WISH, MCF-7 and HeLa cells. Our data clearly show that the two types of interferon have a strong inhibitory effect on the repair of UV-damaged DNA and this effect is cell type-dependent

Copper Accumulation and Phosphatase Activities of Aspergillus and Rhizopus

Abstract Copper accumulation and phosphatase activities of three Aspergillus species resistant to copper were compared to three copper-sensitive Rhizopus species. High level of acid phosphatases and decreased Cu2+-uptake were found with resistant in contrast to sensitive strains. The presence of copper(II) ions in the medium increased the production of acid phosphatases in the resistant A. niger and decreased their activity in the sensitive R. delemar. Copper ions inhibited the activity of A. niger cellular acid phosphatase with a Ki of 8.9x10-4 ᴍ and slightly activated the R. delemar enzyme.

Maximizing early detection of esophageal squamous cell carcinoma via SILAC-proteomics

Commentary to: SILAC-based quantitative proteomic approach to identify potential biomarkers from the esophageal squamous cell carcinoma secretome Manoj Kumar Kashyap, H. C. Harsha, Santosh Renuse, Harsh Pawar, Nandini A. Sahasrabuddhe, Min-Sik Kim, Arivusudar Marimuthu, Shivakumar Keerthikumar, Babylakshmi Muthusamy, Kumaran Kandasamy, Yashwanth Subbannayya, Thottethodi Subrahmanya Keshava Prasad, Riaz Mahmood, Raghothama Chaerkady, Stephen Meltzer, Rekha V. Kumar, Anil K. Rustgi and Akhilesh Pandey

Decreased DNA repair capacity of UV-irradiated cells following interferon treatment.

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-α, IFN-βand IFN-γ on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-α had a stronger inhibitory effect than IFN-γ. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.

Spontaneous Transfection of Mammalian Cells with Plasmid DNA

A simple method for spontaneous transfection into mammalian cells (both adherent and suspension in culture) with plasmid DNA is described. This method does not require any specific DNA carrier or technical device and can be applied for obtaining both transient and stably transfected cells. The efficiency of spontaneous transfection is slightly lower in comparison with that of the conventional calcium phosphate and lipofectin transfection methods and does not depend on the type of cell culture used.