The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFB binding sequences and monitoring tumor necrosis factor ␣-mediated NFB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFB translocation to the nucleus.Because of its easily detected green fluorescence, the green fluorescent protein (GFP) 1 from the jellyfish Aequorea victoria is a widely used reporter in studies of gene expression and protein localization (1-4). GFP fluorescence does not require any substrate or cofactor (5); hence it is possible to use it in many species for live cell detection purposes. The fluorescence of GFPs is dependent on the key sequence Ser-Tyr-Gly (amino acids 65-67). This sequence undergoes spontaneous oxidation to form a cyclized chromophore (6). Enhanced GFP (EGFP) contains mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64 and is encoded by a gene with humanoptimized codons (7-9). Crystallographic structures of wildtype GFP and the mutant S65T reveal that the GFP tertiary structure resembles a barrel (10, 11). GFP is a single chain polypeptide of 238 amino acids (12). Most of these amino acids form  sheets that are compacted through an antiparallel structure to form the barrel. An ␣-helix containing the chromophore is located inside the barrel, which shields it from the external environment. The compact structure makes GFP very stable under a variety of conditions, including treatment with protease (1). The stability of GFP limits its application in some studies, including transcriptional induction studies.Cellular proteins differ widely in their stabilities. Rapid turnover in proteins is often caused by signals that induce protein degradation. In some cases, the signal is a primary sequence such as the PEST sequence, a sequence possibly correlated with protein degradation (13,14)...
We extended our previous GWAS for psoriasis with a a multistage replication study including 8,312 cases and 12,919 controls from China as well as 3,293 cases, 4,188 controls from Germany and the USA, and 254 nuclear families from the USA. We identified 6 new susceptibility loci associated to psoriasis in Chinese, containing candidate genes ERAP1, PTTG1, CSMD1, GJB2, SERPINB8, ZNF816A (PCombined<5×10−8) and replicated one locus 5q33.1 (TNIP1/ANXA6) previously reported (PCombined=3.8×10−21) in European studies. Two of these loci showed evidence for association evidence in the German study, at ZNF816A and GJB2 with P=3.6×10−3 and P=7.9×10−3, respectively. ERAP1 and ZNF816A were preferentially associated with Type I (early onset) psoriasis in Chinese Han population (test for heterogeneity P=6.5×10−3 and P=1.5×10−3, respectively). Comparisons with previous GWAS of psoriasis highlight the heterogeneity of disease susceptibility between Chinese and European populations. Our study identifies new genetic susceptibility factors and suggests new biological pathways in psoriasis.
Photodynamic
therapy (PDT) is a promising strategy for cancer treatment.
It can not only generate reactive oxygen species (ROS) to cause the
chemical damage of tumor cells in the presence of enough oxygen but
also promote the antitumor immunity of T cells through enhancing the
production of interferon γ (IFN-γ). However, one phenomenon
is ignored so far that the enhanced production of IFN-γ caused
by PDT may significantly increase the expression of programmed death-ligand
1 (PD-L1) on the tumor cell membrane and thus could inhibit the immune
killing effects of T cells. Herein, we report the construction of
a composite by loading metformin (Met) and IR775 into a clinically
usable liposome as a two-in-one nanoplatform (IR775@Met@Lip) to solve
this problem. The IR775@Met@Lip could reverse tumor hypoxia to enhance
ROS production to elicit more chemical damage. Besides, the overexpression
of PD-L1 by PDT was also effectively down-regulated. These therapeutic
benefits including decreased PD-L1 expression, alleviated T cell exhaustion,
and reversed tumor hypoxia successfully suppressed both the primary
and abscopal tumor growth in bladder and colon cancers, respectively.
Combining with its excellent biocompatibility, our results indicate
that this IR775@Met@Lip system has great potential to become a highly
effective cancer therapy modality.
The importance of some putative efflux pumps in conferring drug resistance on certain strain of Mycobacterium tuberculosis was determined by using multidrug-resistant (MDR) strain 1499 bearing defined mutations in rpoB and katG. The clinical isolate 1499 was tested for drug sensitivity and expression level of five putative efflux pump genes by real-time RT-PCR. In the presence of rifampicin or isoniazid, this isolate showed increased transcription of putative efflux pump genes Rv1258c and Rv1410c. Rv1819c was overexpressed only upon isoniazid exposure (p<0.05). Overexpression of some efflux genes is associated with multidrug resistance in certain clinical isolate of M. tuberculosis.
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