Polygynous mating impairs body condition and homeostasis in male reindeer (<Emphasis Type="Italic">Rangifer tarandus tarandus</Emphasis>)

Reindeer are polygynous ruminants that breed when plant growth declines in the Arctic. We studied seven males (2 years and older) in two herds with a total of 34 females to describe the costs and consequences of mating or rut. Body mass declined between September and November and did not recover through winter even though food was available ad libitum. Dominance did not affect body mass or any correlate of mass loss, indicating similar costs of rut among males. Males lost 34% of ingesta-free mass in 77 days of rut, which corresponded to depletion of 23% body protein and 78% body lipid. Water flux, plasma insulin, and plasma thyroxine were minimal 23 days after the peak in body mass, indicating low food intake. Maximum plasma testosterone and cortisol also followed peak mass and coincided with the death of two males from acute infections. Loss in body protein did not increase the ratio of urea to creatinine in plasma. Increased variance in plasma osmolality and urea during mass loss indicated altered homeostatic control during rut. Mating compromises survival of males through reduction of body reserves, food intake, and maintenance of tissues. These adverse effects may be the consequence of selection for large body size and aggression in a highly variable competition for mates.Abbreviations BRI body reserve index - T3 tri-iodo-thyronine - U/C urea to creatinine ratio     

pH shift enhancement of Candida utilis pyruvate decarboxylase production

Pyruvate decarboxylase (PDC) catalyses the synthesis of asymmetric carbinols, e.g., chiral precursors for pharmaceuticals such as ephedrine and pseudoephedrine. The production of PDC by Candida utilis in a minimal medium was improved by manipulating the pH during fermentation in a 5 L bioreactor. At an aeration rate of 0.1 vvm with a stirrer speed of 300 rpm at constant pH 6, a specific PDC activity of 141 U/g dry cell weight (DCW) was achieved (average of two fermentations +/-13%). By allowing the yeast to acidify the growth medium from pH 6 to 2.9, the final specific PDC activity increased by a factor of 2.7 to 385 U/g DCW (average from 4 fermentations +/-16%). The effect of this pH drift on PDC production was confirmed by another experiment with a manual shift of pH from 6 to 3 by addition of 5 M sulfuric acid. The final PDC activity was 392 U/g DCW (average from two fermentations +/-5%). However, experiments with constant pH of 6, 5, 4, or 3 resulted in average specific activities of only 102 to 141 U/g DCW, suggesting that a transitional pH change rather than the absolute pH value was responsible for the increased specific PDC activity.     

Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors

Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6-mm deep, stagnant, medium layer covering a cell culture. The measurement set-up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase.     

Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.     

Recent progress in industrial biocatalysis

In recent years, several procedures have been reported for the development of biocatalytic processes. This review focuses on selected examples integrating biocatalysts into a variety of industrially interesting processes ranging from the manufacture of smaller, chiral speciality chemicals to the synthesis of more complex pharmaceutical intermediates. The use of rational protein design, multistep processes and de novo design of enzyme catalysts for the stereocontrolled preparation of important target structures is discussed.     

Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct

A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153A_{M. aq.}-CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l^–1 ω-hydroxy dodecanoic from 10 g l^–1 C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l^–1 ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.     

Cutaneous Collateral Axonal Sprouting Re-Innervates the Skin Component and Restores Sensation of Denervated Swine Osteomyocutaneous Alloflaps

Reconstructive transplantation such as extremity and face transplantation is a viable treatment option for select patients with devastating tissue loss. Sensorimotor recovery is a critical determinant of overall success of such transplants. Although motor function recovery has been extensively studied, mechanisms of sensory re-innervation are not well established. Recent clinical reports of face transplants confirm progressive sensory improvement even in cases where optimal repair of sensory nerves was not achieved. Two forms of sensory nerve regeneration are known. In regenerative sprouting, axonal outgrowth occurs from the transected nerve stump while in collateral sprouting, reinnervation of denervated tissue occurs through growth of uninjured axons into the denervated tissue. The latter mechanism may be more important in settings where transected sensory nerves cannot be re-apposed. In this study, denervated osteomyocutaneous alloflaps (hind- limb transplants) from Major Histocompatibility Complex (MHC)-defined MGH miniature swine were performed to specifically evaluate collateral axonal sprouting for cutaneous sensory re-innervation. The skin component of the flap was externalized and serial skin sections extending from native skin to the grafted flap were biopsied. In order to visualize regenerating axonal structures in the dermis and epidermis, 50um frozen sections were immunostained against axonal and Schwann cell markers. In all alloflaps, collateral axonal sprouts from adjacent recipient skin extended into the denervated skin component along the dermal-epidermal junction from the periphery towards the center. On day 100 post-transplant, regenerating sprouts reached 0.5 cm into the flap centripetally. Eight months following transplant, epidermal fibers were visualized 1.5 cm from the margin (rate of regeneration 0.06 mm per day). All animals had pinprick sensation in the periphery of the transplanted skin within 3 months post-transplant. Restoration of sensory input through collateral axonal sprouting can revive interaction with the environment; restore defense mechanisms and aid in cortical re-integration of vascularized composite allografts.