In this study we have examined the effects of individual gasoline hydrocarbons (C(5-10,12,14) n-alkanes, C(5-8) isoalkanes, alicyclics [cyclopentane and methylcyclopentane] and BTEX compounds [benzene, toluene, ethylbenzene, m-, o-, and p-xylene]) on cometabolism of methyl tertiary butyl ether (MTBE) and tertiary butyl alcohol (TBA) by Mycobacterium austroafricanum JOB5. All of the alkanes tested supported growth and both MTBE and TBA oxidation. Growth on C(5-8) n-alkanes and isoalkanes was inhibited by acetylene whereas growth on longer chain n-alkanes was largely unaffected by this gas. However, oxidation of both MTBE and TBA by resting cells was consistently inhibited by acetylene, irrespective of the alkane used as growth-supporting substrate. A model involving two separate but co-expressed alkane-oxidizing enzyme systems is proposed to account for these observations. Cyclopentane, methylcyclopentane, benzene and ethylbenzene did not support growth but these compounds all inhibited MTBE and TBA oxidation by alkane-grown cells. In the case of benzene, the inhibition was shown to be due to competitive interactions with both MTBE and TBA. Several aromatic compounds (p-xylene > toluene > m-xylene) did support growth and cells previously grown on these substrates also oxidized MTBE and TBA. Low concentrations of toluene (<10 microM) stimulated MTBE and TBA oxidation by alkane-grown cells whereas higher concentrations were inhibitory. The effects of acetylene suggest strain JOB5 also has two distinct toluene-oxidizing activities. These results have been discussed in terms of their impact on our understanding of MTBE and TBA cometabolism and the enzymes involved in these processes in mycobacteria and other bacteria.

The sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) isoform is a promising contraceptive target because it is specific to male germ cells, essential for sperm motility and male fertility, and well suited to pharmacological inhibition. However, GAPDHS is difficult to isolate from native sources and recombinant expression frequently results in high production of insoluble enzyme. We chose to use the Bac-to-Bac baculovirus-insect cell system to express a His-tagged form of human GAPDHS (Hu his-GAPDHS) lacking the proline-rich Nterminal sequence. This recombinant Hu his-GAPDHS was successfully produced in Spodoptera frugiperda 9 (Sf9) cells by infection with recombinant virus as a soluble, enzymatically active form in high yield, >35mg/L culture. Biochemical characterization of the purified enzyme by mass spectrometry and size exclusion chromatography confirmed the presence of the tetrameric form. Further characterization by peptide ion matching mass spectrometry and Edman sequencing showed that unlike the mixed tetramer forms produced in bacterial expression systems, human his-GAPDHS expressed in baculovirus-infected insect cells is homotetrameric. The ability to express and purify active human GAPDHS as homotetramers in high amounts will greatly aid in drug discovery efforts targeting this enzyme for discovery of novel contraceptives and three compounds were identified as inhibitors of Hu his-GAPDHS from a pilot screen of 1120 FDA-approved compounds.

Temperature-triggered copolymers are proposed for a number of bio-applications but there is no ideal material platform, especially for injectable drug delivery. Options are needed for degradable biomaterials that not only respond to temperature but also easily accommodate linkage of active molecules. A first step toward realizing this goal is the design and synthesis of the novel materials reported herein. A multifunctional macromer, methacrylated hyperbranched polyglycerol (HPG-MA) with an average of one acrylate unit per copolymer, was synthesized and copolymerized with N-isopropylacrylamide (NIPAAm), hydroxyethyl methacrylate-polylactide (HEMAPLA) and acrylic acid (AAc). The potential to fully exploit the copolymers by modification of the multiple HPG hydroxyl groups will not be discussed here. Instead, this report focuses on the thermoresponsive, biocompatible, and degradation properties of the material. Poly(NIPAAm-co-HEMAPLA-co-AAc-co-HPG-MA) displayed increasing lower critical solution temperatures (LCST) as the HPG content increased over a range of macromer ratios. For the copolymer with the maximum HPG incorporation (17%), the LCST was ~30 °C. In addition, this sample showed no toxicity when human uterine fibroid cells were co-cultured with the copolymer for up to 72 h. This copolymer lost approximately 92% of its mass after 17 hours at 37 °C. Thus, the reported biomaterials offer attractive properties for the design of drug delivery systems where orthogonally triggered mechanisms of therapeutic release in relatively short time periods would be attractive.

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents.

We have generated a photoactivatable form of sonic hedgehog protein by modifying the N-terminal cysteine with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (Bzm). The Bzm modification on ShhN imparted a significant increase in activity as assessed in the C3H10T1/2 functional assay with potency comparable to that of the endogenous dual-lipidated form of ShhN (ShhNp). Reversed-phase HPLC analysis indicated that the increase in activity compared to unmodified ShhN may be due in part to the hydrophobic nature of the benzophenone group. In contrast to the fully processed ShhNp, Bzm-ShhN is monomeric as assessed by analytical SEC and does not require detergent to be soluble. Further, we demonstrated that the Bzm-ShhN was able to crosslink in vitro in the presence of a known binding partner, heparin. We suggest that Bzm-ShhN can serve as a relatively facile and preferred source of ShhNp for in vitro assays and as a probe to identify novel Hh protein interactions.

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