分子生物学: オープンアクセス

Growth arrest and DNA damage-inducible (GADD) 45 and GADD153 proteins have been implicated in DNA repair, cell cycle regulation and growth arrest along with numerous other cellular mechanisms. In recent years, evidence has emerged that proteins encoded by these genes play pivotal roles in tumor suppression and apoptotic cell death. Thus, compounds altering the expression of these genes are likely to be of interest in cancer prevention and/or treatment. Protocatechualdehyde is isolated from Phellinus gilvus and has been investigated as a promising cancer preventive agent because of its medicinal properties. This mushroom belongs to Hymenochaetaceae Basidiomycetes, and has advantages over many Phellinus species due to short growth period (3 months), making production cost-effective. The exact molecular mechanisms of protocatechualdehyde are not clearly understood. Based on studies of pro-apoptotic activity of protocatechualdehyde in T-cells and colorectal cancer cells, we examined the relationship between the expression of GADD45 and GADD153 and apoptosis induction in human lung cancer cell line PC-9. We report a p⁵³- independent increase in GADD45 and GADD153 expression by protocatechualdehyde. Likewise, the proliferation of PC-9 cells is inhibited via a G1/S arrest of the cell-cycle stimulating apoptosis. Further, induction of apoptosis was inhibited in PC-9 cells knocked down for GADD45 and GADD153. Protocatechualdehyde treatment also induced the expression of cell cycle inhibitors p²¹ and p²⁷, while inhibiting Bcl-2, cyclin D1, CDK2, CDK4 and CDK6 genes. These findings suggest that upregulation of GADD45 and GADD153 proteins are the mechanism for protocatechualdehyde’s anti-tumor activities.

Improved cellular engineering tools have enabled scientists to modify genomic sequences at a new pace for a variety of applications. However, in order to create more accurate tools for site specific targeting and editing, improved delivery systems for modifying enzymes are required. These enzymes allow end users to disrupt, insert or edit genes utilizing sequence specific homology. Current applications require co-transfection of an engineering plasmid in tandem with the vector carrying the modifying enzyme. However, co-transfection can negatively affect transfection efficiencies as well as increase probability of randomly integrating DNA. Here we report the transient delivery of catalyzing enzymes via BacMam non-integrating virus at high efficiency. Baculovirus is a non-infectious delivery system that can transduce a variety of mammalian cells. We have shown improved delivery of NLS-Cre recombinase for the excision of regions in disrupted genes to enable expression. In addition, we have tested PhiC31 integrase delivery for the guided insertion of plasmid DNA to a specific locus. The transient expression provided by BacMam, coupled with ease of transfection, creates an ideal system for delivery of catalyzing enzymes in mammalian cells.

Amylase binding protein A (AbpA) of Streptococcus gordonii serves as a major receptor for human salivary α-amylase (HSAmy), the predominant enzyme in human salivary secretions, to bind to the bacterial cell surface. On enamel surfaces, the binding of AbpA to HSAmy renders S. gordonii act as a scaffold for other oral bacteria to attach to the acquired enamel pellicle leading to complex bacterial communities and invasion of host tissues. While the role of AbpA in adhesion, starch metabolism and biofilm formation in influencing the ecology of the oral biofilms has been established, the structure function relationships of AbpA are yet to be defined. Since distally located aromatic residues of HSAmy are involved in the interaction with AbpA, we hypothesized that AbpA might use separate structural regions to bind to HSAmy. To test this, several deletion mutants of AbpA were constructed and studied to correlate the effect of deletions in binding to HSAmy in the following: 1) their secondary structural features through circular dichroism studies; 2) their ability to alter the capacity of HSAmy to hydrolyze starch and several oligosaccharides after complex formation and 3) their binding to HSAmy using surface plasmon resonance spectroscopy. Our results showed that a) AbpA does not bind at the active site of salivary α-amylase; b) both the N-terminal region (24-56 residues) and a central region (residues 124-165) are required for binding to HSAmy; and c) the C-terminal end residues (166- 195) are not necessary for binding. These results clearly show that AbpA binding to HSAmy encompasses distinct and distal regions of the structure.

Introduction: Rev Responsive Element (RRE) is a RNA molecule responsible to mRNA from HIV-1 virus nuclear transportation to cytoplasm through RRE-Rev pathway, essencial to virus replication. Enfuvirtide resistance mutations are primary located in a perimeter of 10 amino acids of HR1, a corresponded region of RRE. Charactherize RRE should provide a new approach for HIV therapy.

Objectives: Sequence and characterize RRE from gp41 to evaluate variability and correlate with laboratory parameters in sequences from HIV-1-infected patients, whitch were receiving regimens including Enfuvirtide, naïve or rescue therapy.

Methods: Sixty-two samples from HIV patients in Northeast Brazil were collected and Thirty-five RRE sequences and clinical follow-up were analyzed, distributed into three groups: N (naïve therapy), T (treated patients with rescue regimens) and F (rescue regimens containing Enfuvirtide). Sequences obtained were aligned with Los Alamos HIV sequence database by using the HIV BLAST Search.

Results: A phylogenetic analyses demonstrated higher prevalence of HIV-1 subtypes B (97.2%). An increased immunology response was observed in CD4 count higher on group T (71,5%) compared with F (2,98%). Group N most commum mutations and polymorphisms were Q32L (41.6%), N42S (8.3%), R46K (33.3%), L54M (41.6%); group T: Q32R (8.3%), R46K (25%), L54M (33.3%); and group F: Q32L (18.2%), G36D (9.1%), V38A (9.1%), N42S (27.3%), N42T (9.1%), R46K (27.3%), L54M (45.4%), K77R (54.5%). Three samples demonstrated significant resistance mutations to fusion inhibitors. Analysis of RRE nucleotide primary sites observed mutation 28A in 27.2% and 8.3% on groups F and N respectively, and 27S in 8.3% on group T. There was selective pressure on HR1 region from HIV-1 patients using antiretrovirals, independent of enfuvirtide exposure.

Conclusions: This study defined most prevalent RRE polymorphisms in Northeast Brazil and suggests highly preserved regions primary sites to Rev connection. Observed a low resistance profile to enfuvirtide in failing regimens with this drug. Selective pressure on HR1 region in failed regimens with out fusion inhibidors was detected.