Minimal Erythema Dose (MED) Testing

Ultraviolet radiation (UV) therapy is sometimes used as a treatment for various common skin conditions, including psoriasis, acne, and eczema. The dosage of UV light is prescribed according to an individual''s skin sensitivity. Thus, to establish the proper dosage of UV light to administer to a patient, the patient is sometimes screened to determine a minimal erythema dose (MED), which is the amount of UV radiation that will produce minimal erythema (sunburn or redness caused by engorgement of capillaries) of an individual''s skin within a few hours following exposure. This article describes how to conduct minimal erythema dose (MED) testing. There is currently no easy way to determine an appropriate UV dose for clinical or research purposes without conducting formal MED testing, requiring observation hours after testing, or informal trial and error testing with the risks of under- or over-dosing. However, some alternative methods are discussed.     

Effects of biocontrol agents on lipid and protein composition of Indian mustard seeds from plants infected with Alternaria species

Field experiments were carried out with Indian mustard (Brassica juncea L. Cv RLM 1359) to investigate the influence of biocontrol agents on seeds from plants infected with Alternaria blight. The biocontrol agents viz, Trichoderma harzianum, Pseudomonas fluorescens and Bacillus subtilis were applied as seed treatment/seed treatment coupled with spray on 30 and 60 days after sowing of seeds in experimental fields. The plants treated with different biocontrol agents were more developed than non-treated plants throughout the experiment. Biochemical analysis revealed that application of biocontrol agents resulted in increase in lipid and protein content in seeds from treated plants. The proportion of various lipidic fractions i.e. phospholipids, glycolipids and sterol content in seeds increased with a corresponding decrease in total glycerides. The proportion of 18:3, 20:1 and 22:1 fatty acids increased while that of 18:1 and 18:2 fatty acids decreased in seeds with application of biocontrol agents. There were both qualitative and quantitative differences in the banding patterns of albumin and globulin proteins after application of biocontrol agents. The data suggested that biochemical alterations in the host induced by treatment with biocontrol agents could be associated with defence mechanisms and enhanced growth of the plant.     

Evidence for a causal role of CD38 expression in granulocytic differentiation of human HL-60 cells

Granulocytic differentiation of human HL-60 cells can be induced by retinoic acid and is accompanied by a massive expression of CD38, a multi-functional enzyme responsible for metabolizing cyclic ADP-ribose (cADPR), a Ca(2+) messenger. Immunofluorescence staining showed that CD38 was expressed not only on the surface of intact HL-60 cells but also intracellularly, which was revealed after permeabilization with Triton. Concomitant with CD38 expression was the accumulation of cADPR, and both time courses preceded the onset of differentiation, suggesting a causal role for CD38. Consistently, treatment of HL-60 cells with a permeant inhibitor of CD38, nicotinamide, inhibited both the CD38 activity and differentiation. More specific blockage of CD38 expression was achieved by using morpholino antisense oligonucleotides targeting its mRNA, which produced a corresponding inhibition of differentiation as well. Similar inhibitory effects were observed when CD38 expression was reduced by the RNA interference technique targeting two separate regions of the coding sequence of CD38. Further support came from transfecting HL-60 cells with a Tet-On expression vector containing a full-length CD38. Subsequent treatments with doxycycline induced both CD38 expression and differentiation in the absence of retinoic acid. These results provide the first evidence that CD38 expression and the consequential accumulation of cADPR play a causal role in mediating cellular differentiation.     

Proteinase suppression by E-cadherin-mediated cell-cell attachment in premalignant oral keratinocytes

The expression and activity of epithelial proteinases is under stringent control to prevent aberrant hydrolysis of structural proteins and disruption of tissue architecture. E-cadherin-dependent cell-cell adhesion is also important for maintenance of epithelial structural integrity, and loss of E-cadherin expression has been correlated with enhanced invasive potential in multiple tumor models. To address the hypothesis that there is a functional link between E-cadherin and proteinase expression, we have examined the role of E-cadherin in proteinase regulation. By using a calcium switch protocol to manipulate junction assembly, our data demonstrate that initiation of de novo E-cadherin-mediated adhesive contacts suppresses expression of both relative matrix metalloproteinase-9 levels and net urinary-type plasminogen activator activity. E-cadherin-mediated cell-cell adhesion increases both phosphatidylinositol 3'-kinase (PI3-kinase)-dependent AKT phosphorylation and epidermal growth factor receptor-dependent MAPK/ERK activation. Pharmacologic inhibition of the PI3-kinase pathway, but not the epidermal growth factor receptor/MAPK pathway, prevents E-cadherin-mediated suppression of proteinases and delays junction assembly. Moreover, inhibition of junction assembly with a function-blocking anti-E-cadherin antibody stimulates proteinase-dependent Matrigel invasion. As matrix metalloproteinase-9 and urinary-type plasminogen activator potentiate the invasive activity of oral squamous cell carcinoma, these data suggest E-cadherin-mediated signaling through PI3-kinase can regulate the invasive behavior of cells by modulating proteinase secretion.     

Biochemical characterization of the Staphylococcus aureus PcrA helicase and its role in plasmid rolling circle replication

Previous genetic studies have suggested that a putative chromosome-encoded helicase, PcrA, is required for the rolling circle replication of plasmid pT181 in Staphylococcus aureus. We have overexpressed and purified the staphylococcal PcrA protein and studied its biochemical properties in vitro. Purified PcrA helicase supported the in vitro replication of plasmid pT181. It had ATPase activity that was stimulated in the presence of single-stranded DNA. Unlike many replicative helicases, PcrA was highly active as a 5' --> 3' helicase and had a weaker 3' --> 5' helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of replication and becomes covalently attached to the 5' end of the DNA. The 3' OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a protein-protein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.     

c-Src mediates mitogenic signals and associates with cytoskeletal proteins upon vascular endothelial growth factor stimulation in Kaposi's sarcoma cells

Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi's sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.     

Phenotypic and functional effects of heat shock protein 90 inhibition on dendritic cell

The 90-kDa heat shock protein (Hsp90) plays an important role in conformational regulation of cellular proteins and thereby cellular signaling and function. As Hsp90 is considered a key component of immune function and its inhibition has become an important target for cancer therapy, we here evaluated the role of Hsp90 in human dendritic cell (DC) phenotype and function. Hsp90 inhibition significantly decreased cell surface expression of costimulatory (CD40, CD80, CD86), maturation (CD83), and MHC (HLA-A, B, C and HLA-DP, DQ, DR) markers in immature DC and mature DC and was associated with down-regulation of both RNA and intracellular protein expression. Importantly, Hsp90 inhibition significantly inhibited DC function. It decreased Ag uptake, processing, and presentation by immature DC, leading to reduced T cell proliferation in response to tetanus toxoid as a recall Ag. It also decreased the ability of mature DC to present Ag to T cells and secrete IL-12 as well as induce IFN-gamma secretion by allogeneic T cells. These data therefore demonstrate that Hsp90-mediated protein folding is required for DC function and, conversely, Hsp90 inhibition disrupts the DC function of significant relevance in the setting of clinical trials evaluating novel Hsp90 inhibitor therapy in cancer.