Inhibitory effects of substrate and product on the carvone biotransformation activity of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.     

The structure and orientation of the C-terminus of LRAP

Amelogenin is the predominant protein found during enamel development and is thought to be the biomineralization protein controlling the unique elongated hydroxyapatite crystals that constitute enamel. The secondary structure of biomineralization proteins is thought to be important in the interaction with hydroxyapatite. Unfortunately, very little data are available on the structure or the orientation of amelogenin, either in solution or bound to hydroxyapatite. The C-terminus contains the majority of the charged residues and is predicted to interact with hydroxyapatite; thus, we used solid-state NMR dipolar recoupling techniques to investigate the structure and orientation of the C-terminus of LRAP, a naturally occurring splice variant of full-length amelogenin. Using ¹³C{¹⁵N} Rotational Echo DOuble Resonance (REDOR), the structure of the C-terminus was found to be largely random coil, both on the surface of hydroxyapatite as well as lyophilized from solution. The orientation of the C-terminal region with respect to hydroxyapatite was investigated for two alanine residues (Ala⁴⁶ and Ala⁴⁹) using ¹³C{³¹P} REDOR and one lysine residue (Lys⁵²) using ¹⁵N{³¹P} REDOR. The residues examined were found to be 7.0, 5.7, and 5.8 Å from the surface of hydroxyapatite for Ala⁴⁶, Ala⁴⁹, and Lys⁵², respectively. This provides direct evidence that the charged C-terminus is interacting closely with hydroxyapatite, positioning the acidic amino acids to aid in controlling crystal growth. However, solid-state NMR dynamics measurements also revealed significant mobility in the C-terminal region of the protein, in both the side chains and the backbone, suggesting that this region alone is not responsible for binding.     

Improved reactor performance and operability in the biotransformation of carveol to carvone using a solid-liquid two-phase partitioning bioreactor

In an effort to improve reactor performance and process operability, the microbial biotransformation of (-)-trans-carveol to (R)-(-)-carvone by hydrophobic Rhodococcus erythropolis DCL14 was carried out in a two phase partitioning bioreactor (TPPB) with solid polymer beads acting as the partitioning phase. Previous work had demonstrated that the substrate and product become inhibitory to the organism at elevated aqueous concentrations and the use of an immiscible second phase in the bioreactor was intended to provide a reservoir for substrates to be delivered to the aqueous phase based on the metabolic rate of the cells, while also acting as a sink to uptake the product as it is produced. The biotransformation was previously undertaken in a two liquid phase TPPB with 1-dodecene and with silicone oil as the immiscible second phase and, although improvement in the reactor performance was obtained relative to a single phase system, the hydrophobic nature of the organism caused the formation of severe emulsions leading to significant operational challenges. In the present work, eight types of polymer beads were screened for their suitability for use in a solid-liquid TPPB for this biotransformation. The use of selected solid polymer beads as the second phase completely prevented emulsion formation and therefore improved overall operability of the reactor. Three modes of solid-liquid TPPB operation were considered: the use of a single polymer bead type (styrene/butadiene copolymer) in the reactor, the use of a mixture of polymer beads in the reactor (styrene/butadiene copolymer plus Hytrel(R) 8206), and the use of one type of polymer beads in the reactor (styrene/butadiene copolymer), and another bead type (Hytrel(R) 8206) in an external column through which fermentation medium was recirculated. This last configuration achieved the best reactor performance with 7 times more substrate being added throughout the biotransformation relative to a single aqueous phase benchmark reactor and 2.7 times more substrate being added relative to the best two liquid TPPB case. Carvone was quantitatively recovered from the polymer beads via single stage extraction into methanol, allowing for bead re-use.     

Promoting More Modest Weight Losses: A Pilot Study

Objective: This pilot study assessed the short‐ and long‐term effects of a modified cognitive behavioral treatment designed to facilitate obese patients’ acceptance of a 5% to 10% reduction in initial weight. Research Methods and Procedures: Participants were 17 women with a mean age of 46.5 ± 9.7 years and BMI of 34.7 ± 2.9 kg/m². They participated in a 40‐week program that included four phases. The first discussed the benefits of modest weight losses and the potential adverse effects of unrealistic expectations. Phase II provided instruction in traditional cognitive behavioral methods of weight control Phase III focused on methods to improve body image and self‐esteem. Phase IV addressed skills for weight maintenance. Changes in weight, self‐esteem, body image, and quality of life were assessed at the end of treatment and 1 year later (week 92). Results: At week 40, participants lost an average of 5.7 ± 5.3% of initial weight, which was associated with significant improvements in body image, self‐esteem, and quality of life. Improvements in psychosocial status were maintained at week 92, although mean weight loss at this time had declined to 2.9 ± 5.6% of initial weight. Increased satisfaction with body weight at week 40 was associated with significantly better maintenance of weight loss at follow‐up (r = ?0.70; p = 0.02). Discussion: Having participants seek only modest initial weight losses does not appear to facilitate weight maintenance. However, increasing patients’ satisfaction with their body weight at the end of treatment may help improve weight maintenance. More research is needed on the relation between satisfaction with initial weight loss and long‐term success.     

Rod Microglia: A Morphological Definition

Brain microglial morphology relates to function, with ramified microglia surveying the micro-environment and amoeboid microglia engulfing debris. One subgroup of microglia, rod microglia, have been observed in a number of pathological conditions, however neither a function nor specific morphology has been defined. Historically, rod microglia have been described intermittently as cells with a sausage-shaped soma and long, thin processes, which align adjacent to neurons. More recently, our group has described rod microglia aligning end-to-end with one another to form trains adjacent to neuronal processes. Confusion in the literature regarding rod microglia arises from some reports referring to the sausage-shaped cell body, while ignoring the spatial distribution of processes. Here, we systematically define the morphological characteristics of rod microglia that form after diffuse brain injury in the rat, which differ morphologically from the spurious rod microglia found in uninjured sham. Rod microglia in the diffuse-injured rat brain show a ratio of 1.79±0.03 cell length∶cell width at day 1 post-injury, which increases to 3.35±0.05 at day 7, compared to sham (1.17±0.02). The soma length∶width differs only at day 7 post-injury (2.92±0.07 length∶width), compared to sham (2.49±0.05). Further analysis indicated that rod microglia may not elongate in cell length but rather narrow in cell width, and retract planar (side) processes. These morphological characteristics serve as a tool for distinguishing rod microglia from other morphologies. The function of rod microglia remains enigmatic; based on morphology we propose origins and functions for rod microglia after acute neurological insult, which may provide biomarkers or therapeutic targets.