A rapid radiochemical filter paper assay for determination of hexokinase activity and affinity for glucose-6-phosphate

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses, and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as a substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and KM. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions

Constitutively active ErbB2 regulates cisplatin-induced cell death in breast cancer cells via pro- and antiapoptotic mechanisms

Despite the frequent expression of N-terminally truncated ErbB2 (DNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how DNErbB2 affects chemotherapy-induced cell death. Previously it was shown that DNErbB2 upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Naþ/Hþ exchanger (SLC9A1/NHE1) strongly sensitizes DNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which DNErbB2 regulates cisplatin-induced breast cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9,-7, fodrin, and PARP-1 inMCF-7 cells. Inducible DNErbB2 expres-sion strongly reduced cisplatin-induced ATM- andp53-phosphor-ylation, augmented Noxa upregulation and caspase-9 and-7 cleavage, doubled p21WAF1/Cip1 (CDKN1A) expression, and near-ly abolished Bcl-2 expression. LC3-GFP analysis demonstrated that autophagic flux was reduced by cisplatin in a manner aug-mented by DNErbB2, yet did not contribute to cisplatin-induced death. Using knockdown approaches, it was shown that cisplatin-induced caspase-7 cleavage in DNErbB2-MCF-7 cells was Noxa-and caspase-9 dependent. This pathwaywas augmented byNHE1 inhibition, while the Naþ/HCO3 cotransporter (SLC4A7/ NBCn1) was internalized following cisplatin exposure. Implications: This work reveals that DNErbB2 strongly affects several major pro- and antiapoptotic pathways and provides mechanistic insight into the role of NHE1 in chemotherapy resistance. These findings have relevance for defining therapy regimens in breast cancers with DNErbB2 and/or NHE1 over-expression. Mol Cancer Res; 13(1); 63–77. 2014 AACR

Stratum Corneum Lipids in Non-Lesional Atopic and Healthy Skin following Moisturizer Application: A Randomized Clinical Experiment

Introduction: It is an international standard to recommend patients with atopic dermatitis (AD) to use moisturizers; however, little is known about their effect on lipids in the stratum corneum (SC). Methods: In this randomized clinical experiment of 30 Caucasian participants (15 with AD and 15 healthy controls), the superficial SC lipid profile was assessed through tape stripping non-lesional skin following treatment thrice daily for seven days with a moisturizer, and subsequently compared with untreated skin. Results: No discernible disparity in superficial SC lipid quantity was evident between the AD group and the control group. However, the SC lipid composition diverged significantly, with the AD group exhibiting diminished levels of long-chain EO CERs (p = 0.024) and elevated levels of short-chain C34 CERs (p = 0.025) compared to healthy skin. Moisturizer application significantly reduced the total SC lipids and all lipid subgroups in both groups. Within the AD group, a non-significant inclination towards an augmentation in EO CERs (p = 0.053) and reduction in C34 CERs (p = 0.073) was observed. Conclusion: The recent identification of distinctions in SC lipid composition between AD and healthy skin was substantiated by our findings. Topical moisturizer application, despite reducing overall total lipids, indicated a potential tendency towards a healthier lipid constitution in AD skin

Skin dysbiosis in the microbiome in atopic dermatitis is site-specific and involves bacteria, fungus and virus

BACKGROUND: Microbial dysbiosis with increased Staphylococcus aureus (S. aureus) colonization on the skin is a hallmark of atopic dermatitis (AD), however most microbiome studies focus on bacteria in the flexures and the microbial composition at other body sites have not been studied systematically. OBJECTIVES: The aim of the study is to characterize the skin microbiome, including bacteria, fungi and virus, at different body sites in relation to AD, lesional state, and S. aureus colonization, and to test whether the nares could be a reservoir for S. aureus strain colonization. METHODS: Using shotgun metagenomics we characterized microbial compositions from 14 well defined skin sites from 10 patients with AD and 5 healthy controls. RESULTS: We found clear differences in microbial composition between AD and controls at multiple skin sites, most pronounced on the flexures and neck. The flexures exhibited lower alpha-diversity and were colonized by S. aureus, accompanied by S. epidermidis in lesions. Malassezia species were absent on the neck in AD. Virus mostly constituted Propionibacterium and Staphylococcus phages, with increased abundance of Propionibacterium phages PHL041 and PHL092 and Staphylococcus epidermidis phages CNPH82 and PH15 in AD. In lesional samples, both the genus Staphylococcus and Staphylococcus phages were more abundant. S. aureus abundance was higher across all skin sites except from the feet. In samples where S. aureus was highly abundant, lower abundances of S. hominis and Cutibacterium acnes were observed. M. osloensis and M. luteus were more abundant in AD. By single nucleotide variant analysis of S. aureus we found strains to be subject specific. On skin sites some S. aureus strains were similar and some dissimilar to the ones in the nares. CONCLUSIONS: Our data indicate a global and site-specific dysbiosis in AD, involving both bacteria, fungus and virus. When defining targeted treatment clinicians should both consider the individual and skin site and future research into potential crosstalk between microbiota in AD yields high potential. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-021-02302-2