Multiple global suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM β-lactamases

The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

SF20/IL-25, a novel bone marrow stroma-derived growth factor that binds to mouse thymic shared antigen-1 and supports lymphoid cell proliferation.

Using a forward genetic approach and phenotype-based complementation screening to search for factors that stimulate cell proliferation, we have isolated a novel secreted bone marrow stroma-derived growth factor, which we termed SF20/IL-25. This protein signals cells to proliferate via its receptor, which we have identified as mouse thymic shared Ag-1 (TSA-1). Enforced expression of TSA-1 in IL-3-dependent Ba/F3 cells that do not express endogenous TSA-1 rendered cells to proliferate in a dose-dependent manner when stimulated with SF20/IL-25. FDCP2, a factor-dependent hemopoietic cell line that expresses endogenous TSA-1, could also be stimulated to proliferate with SF20/IL-25. Binding of SF20 to TSA-1 was blocked by anti-TSA-1 Ab and SF20-induced proliferation of TSA-1-expressing cells was inhibited by anti-TSA-1. In vitro assay revealed that SF20/IL-25 has no detectable myelopoietic activity but supports proliferation of cells in the lymphoid lineage.     

A novel method for quantifying cell migration through tissue engineering scaffolds: evaluation of modified collagen matrices

A novel method to quantify cell migration through potential tissue engineering 3-d scaffolds is described. The migration assay uses a dot-blotting apparatus into which the tissue engineering matrix is placed on top of a nitrocellulose membrane. This assay was used to evaluate human dermal fibroblast migration through four porcine collagen matrices with varying pore diameters and pitch lengths. Fibroblasts were placed on the matrix surface, at between 1 ×10³–3 × 10³ cells mm^–2, and left for 18 h to allow migration. The nitrocellulose membrane was stained with haematoxylin, the membrane digitised and the pixel intensity of the stained cells quantified. We showed that for all matrix variants, migration was more effective with a higher initial seeding density. The application of varying initial cell densities resulted in the greatest extent of cell migration through the matrix variant with pores of 30 m diameter and 400 m pitch length (i.e. 10.3% migration at 1 ×10³ cells mm^–2). This method was coupled with confocal microscopy to evaluate the depth of cell migration within the matrix. At a depth of 20 m cell numbers were similar to those on the matrix surface: at a depth of 100 m only a few cells were observed.     

Permeability of the blood-brain barrier to a novel satiety molecule nesfatin-1

Nesfatin-1 has recently been identified as a hypothalamic and brain stem peptide that regulates feeding behavior. Here, we determined the ability of nesfatin-1 to cross the blood–brain barrier (BBB) of mice. We used multiple-regression analysis to determine that radioactively labeled nesfatin-1 injected intravenously entered the brain. The entry rate (K_i) of ¹³¹I-nesfatin-1 from blood-to-brain was 0.20 ± 0.02 μl/g min. This modest rate of entry was not inhibited by the administration of nonradioactive nesfatin-1, suggesting that BBB transport of nesfatin-1 into the brain is by a nonsaturable mechanism. High performance liquid chromatography (HPLC) and acid precipitation showed that most of the injected radiolabeled nesfatin-1 reached the brain as intact peptide, and capillary depletion with vascular washout revealed that 67% of ¹³¹I-nesfatin-1 crossed the BBB to reach the brain parenchyma. Efflux of labeled nesfatin-1 from brain back into blood was by way of bulk flow. These findings demonstrate that nesfatin-1 crosses the BBB in both the blood-to-brain and brain-to-blood directions by nonsaturable mechanisms.