Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.     

Measurement of pancreatic islet cell proliferation by heavy water labeling

We describe a sensitive technique for measuring long-term islet cell proliferation rates in vivo in rats. Pancreatic islets were isolated and the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of DNA was measured by GC-MS. The results of heavy water labeling and BrdU staining were compared. The two methods were highly correlated (r = 0.9581, P < 0.001). Based on long-term heavy water labeling, approximately 50% of islet cells divided in rats between 8 and 15 wk of age. Of interest, long-term BrdU administration suppressed proliferation of islet cells significantly, but not of bone marrow cells. Physiological evidence further supported the validity of the method: older animals (24 wk old) had 60% lower islet cell proliferation rates than younger rats (5 wk old), and partial (50%) pancreatectomy increased proliferation by 20%. In addition, cholecystokinin-8 treatment significantly stimulated proliferation in pancreatectomized rats only. In summary, heavy water labeling is a quantitative approach for measuring islet cell proliferation and testing therapeutic agents.     

An Approaching Motor Boat Induces Stress-Related Behaviors in Proboscis Monkeys (Nasalis larvatus) Living in a Riparian Area

International Journal of Primatology - Primate ecotourism is a fast-growing tourism sector that may have a negative effect on wildlife. In riparian areas, tourists can conveniently reach primates...     

Tannic Acid Attenuates Peripheral and Brain Changes in a Preclinical Rat Model of Glioblastoma by Modulating Oxidative Stress and Purinergic Signaling

Neurochemical Research - Glioblastoma (GB) is a highly aggressive and invasive brain tumor; its treatment remains palliative. Tannic acid (TA) is a polyphenol widely found in foods and possesses...     

Novel meriolin derivatives as rapid apoptosis inducers

3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising derivatives were identified as apoptosis inducers and, emphasizing the multikinase inhibition potential, as sphingosine kinase 2 inhibitors. Our measurements provide additional insights into the structure-activity relationship of meriolin derivatives, suggesting derivatives bearing a pyridine moiety with amino groups in 2-position as most active anticancer compounds and thus as highly promising candidates for future in vivo studies.     

Structural and Functional Analyses of the Human PDH Complex Suggest a “Division-of-Labor” Mechanism by Local E1 and E3 Clusters

1. Download : Download high-res image (274KB)
2. Download : Download full-size image

     

EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5′-3′ exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.     

Effect of physical weathering on the carbon sequestration potential of biochars and hydrochars in soil

Physical weathering can modify the stability of biochar after field exposure. The aim of our study was to determine the potential carbon sequestration of the two chars at different timescales. We investigated the modification in composition and stability resulting from physical weathering of two different chars produced (i) at low temperature (250 °C) by hydrothermal carbonization (HTC); and (ii) at high temperature (1200 °C) by gasification (GS) using contrasting feedstocks. Physical weathering of HTC and GS placed on a water permeable canvas was performed through successive wetting/drying and freezing/thawing cycles. Carbon loss was assessed by mass balance. Chemical stability of the remaining material was evaluated as resistance to acid dichromate oxidation, and biological stability was assessed during laboratory incubation. Moreover, we assessed modification in potential priming effects due to physical weathering. Physical weathering induced a carbon loss ranging between 10 and 40% of the total C mass depending on the feedstock. This C loss is most probably related to leaching of small particulate and dissolved compounds. GS produced from maize silage showed the highest C loss. The chemical stability of HTC and GS was unaffected by physical weathering. In contrast, physical weathering strongly increased the biological stability of HTC and GS char produced from maize silage. After physical weathering, the half‐life (t_1/2) of GS was doubled but only slight increase was noted for those of HTC. During the first weeks of incubation, HTC addition to soil stimulated native soil organic matter (SOM) mineralization (positive priming effect), while the GS addition led to protection of the native SOM against biologic degradation (negative priming effect). Physical weathering led to reduction in these priming effects. Model extrapolations based on our data showed that decadal C sequestration potential of GS and HTC is globally equivalent when all losses including those due to priming and physical weathering were taken into account. However, at century scale only GS may have the potential to increase soil C storage.