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Lung cancer is a major health challenge worldwide. Gefitinib, a tyrosine kinase inhibitor (TKI), is the common therapeutic drug used in advanced non-small-cell lung cancer (NSCLC). However, it is eventually bound to face the problem of acquired drug resistance. In this work, we investigated the role of lncRNA MITA1 in the acquisition of gefitinib resistance in NSCLC and uncovered the possible underlying molecular mechanism of the same. Experiments were carried out using the HCC827 and HCC827GR cells. These were transfected with pcDNA-MITA1 or si-MITA1 and treated with gefitinib. Subsequently, lncRNA MITA1 mediated effect on cell viability and apoptosis were studied using the MTT and flow cytometry assays. Furthermore, using qRT-PCR, Western blotting, and immunofluorescence assays, the regulatory association between lncRNA MITA1 and markers of autophagy (LC3, Beclin-1, and p62) were examined by estimating their cellular protein levels. Also, these results were verified in the presence of an autophagy inhibitor bafilomycin A1. We found that MITA1 was highly upregulated in the gefitinib-resistant NSCLC cells, indicating the regulatory role of MITA1 in gefitinib resistance. Mechanistically, upregulated MITA1 led to gefitinib resistance by suppressing apoptosis, increasing cell viability, and inducing autophagy. Furthermore, these results were true when tested in the presence of bafilomycin A1. Our results suggest that MITA1 by inducing autophagy could be a key regulator of gefitinib resistance in NSCLC.

Malignant glioma is the most common primary brain tumor. Malignant melanoma is the most malignant of skin tumor. The two malignancies are poorly responsive to conventional treatment regimens such as chemotherapy. Temozolomide (TMZ) is a DNA-alkylating agent used for the treatment of glioma, astrocytoma, and melanoma. Resistance to alkylating agents such as TMZ correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Several studies in animal models have demonstrated that decreasing MGMT level with gene therapy could overcome TMZ resistance and enhance tumor cell death. In the present review, we provide an overview of recent advances in this field.

We report here the clinical, genetic, and molecular characterization of three Chinese families with maternally transmitted Leber's hereditary optic neuropathy (LHON). Clinical and genetic evaluations revealed the variable severity and age-of-onset in visual impairment in these families. In the affected matrilineal relatives, the loss of central vision is bilateral, the fellow eye becoming affected either simultaneously (45%) or sequentially (55%). The penetrances of vision loss in these pedigrees were 27%, 50%, and 60%, respectively. The age-at-onset of vision loss in these families was 14, 19, and 24 years, respectively. Furthermore, the ratios between affected male and female matrilineal relatives were 1:1, 1:1.2, and 1:2, respectively. Mutational analysis of mitochondrial DNA revealed the presence of homoplasmic ND6 T14484C mutation, which has been associated with LHON. The incomplete penetrance and phenotypic variability implicate the involvement of nuclear modifier gene(s), environmental factor(s) or mitochondrial haplotype(s) in the phenotypic expression of the LHON-associated T14484C mutation in these Chinese pedigrees.

The 3C-like proteinase of severe acute respiratory syndrome coronavirus (SARS) has been proposed to be a key target for structural based drug design against SARS. We have designed and synthesized 34 peptide substrates and determined their hydrolysis activities. The conserved core sequence of the native cleavage site is optimized for high hydrolysis activity. Residues at position P4, P3, and P3' are critical for substrate recognition and binding, and increment of beta-sheet conformation tendency is also helpful. A comparative molecular field analysis (CoMFA) model was constructed. Based on the mutation data and CoMFA model, a multiply mutated octapeptide S24 was designed for higher activity. The experimentally determined hydrolysis activity of S24 is the highest in all designed substrates and is close to that predicted by CoMFA. These results offer helpful information for the research on the mechanism of substrate recognition of coronavirus 3C-like proteinase.

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.

Human adamalysin 19 (hADAM19)/meltrin β is a member of the ADAM (a disintegrin and metalloproteinase) family and an active metalloproteinase. It is a new metalloproteinase and disintegrin dendritic cell antigen marker. Adamalysin 19 gene was expressed in normal and transformed tissues and cells such as placenta, brain, heart, leukocytes, and colorectal adenocarcinoma SW480. To develop specific tools to investigate the functions of hADAM19, peptide antigens were rationally selected and specific polyclonal antibodies (pAbs) were developed to modulate hADAM19 activity. Anti-metalloproteinase and anti-disintegrin domain IgG molecules inhibited the α-2-macroglobulin cleavage by hADAM19; however, their pre-immune and anti-pro-domain IgG molecules did not. Since anti-disintegrin IgG also neutralized the proteolytic activity, the disintegrin domain may affect the hADAM19 protein folding and/or substrate binding. These pAbs may be used to specifically localize the hADAM19 protein in tissues and cells and elucidate its biological and pathological functions such as processing pro-growth factors.

The adamalysins are involved in proteolysis, adhesion, fusion, and intracellular signaling. Human ADAM19/adamalysin-19 (A disintegrin and metalloproteinase 19) was identified from primary dendritic cell cDNA libraries. It has a signal sequence, a pro-domain with a “cysteine-switch” residue, a metalloproteinase domain with a zinc-binding site, a disintegrin, a cysteine-rich domain, an epidermal-growth-factor-like domain, a transmembrane domain, and a cytoplasmic domain with putative SH3 ligand binding sites. Its mRNA was expressed in the placenta, heart, bladder, lymph nodes, and leukocytes, colorectal adenocarcinoma SW 480, and other organs/cells. The hADAM19 recombinant protein was expressed in human cells. It formed a complex with and cleaved alpha-2 macroglobulin (α2-M). Its proteolytic activity was blocked by 1,10-phenanthroline, EDTA, EGTA, and a synthetic matrix metalloproteinase (MMP) inhibitor and not by the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. It did not cleave the MMP substrates tested, e.g., type I collagen and gelatin, casein, and four peptide substrates. Thus, hADAM19 is an active metalloproteinase and may have a specific substrate profile.

We report here the clinical, genetic, and molecular characterization of three Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age of onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T14502C (I58V) mutation, which localized at a highly conserved isoleucine at position 58 of ND6, and distinct sets of mtDNA polymorphisms belonging to haplogroups M10a, F1a1, and H2. The occurrence of T14502C mutation in these several genetically unrelated subjects affected by visual impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. Here, mtDNA variants I187T in the ND1, A122V in CO1, S99A in the A6, and V254I in CO3 exhibited an evolutionary conservation, indicating a potential modifying role in the development of visual impairment associated with T14502C mutation in those families. Furthermore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic manifestation of the LHON-associated T14502C mutation in these Chinese families.

We report here the clinical, genetic and molecular characterization of four Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age-of-onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T3394C (Y30H) mutation, which localized at a highly conserved tyrosine at position 30 of ND1, and distinct sets of mtDNA polymorphisms belonging to haplogroups D4b and M9a. The occurrence of T3394C mutation in these several genetically unrelated subjects affected by visual impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. However, there was the absence of functionally significant mtDNA mutations in these four Chinese pedigrees carrying the T3394C mutation. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated T3394C mutation.

A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1−/− mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1−/− yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1−/− embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.

We report here the clinical, genetic and molecular characterization of one three-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON) and hearing loss. Four of 14 matrilineal relatives exhibited the moderate central vision loss at the average age of 12.5 years. Of these, one subject exhibited both LHON and mild hearing impairment. Sequence analysis of the complete mitochondrial genomes in the pedigree showed the presence of homoplasmic LHON-associated ND6 T14484C mutation, deafness-associated 12S rRNA A1555 mutation and 47 other variants belonging to Eastern Asian haplogroup H2. None of other mitochondrial variants was evolutionarily conserved and functional significance. Therefore, the coexistence of the A1555G mutation and T14484C mutations in this Chinese family indicate that the A1555G mutation may play a synergistic role in the phenotypic manifestation of LHON associated ND6 T14484C mutation. However, the incomplete penetrance of vision and hearing loss suggests the involvement of nuclear modifier genes and environmental factors in the phenotypic expression of these mtDNA mutations.