Interaction of detergents with bovine lens α-crystallin: evidence for an oligomeric structure based on amphiphilic interactions

We have studied the quaternary structure of α-crystallin in the presence of increasing concentrations of amphiphilic and neutral detergents using gel filtration, light-scattering, boundary and equilibrium sedimentation. We observed a continuous reduction of the molar mass of the polymeric α-crystallin on increasing the concentration of sodium dodecyl sulphate from 0.1 mM to 5 mM, ending up with the monomeric peptides. Dodecyltrimethylammonium bromide also disrupts the oligomeric structure of α-crystallin but the interaction appears to be cooperative: in the sharp transition region (for a 1 mg/ml protein solution) from 3 to 8 mM of the detergent, only the native protein and a mixture of monomeric and dimeric peptide-DTAB complexes can be observed. Concomitant studies of the circular dichroism in the far UV revealed a substantial decrease of the β-sheet and increase of the α-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent αA and αB peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the αA and αB peptides and the formation of the oligomeric α-crystallin structure: the binding of the amphiphilic detergent reduces the β-sheet content, induces the formation of α-helix secondary structure and reduces the tendency of the peptide to form large aggregates. The different mechanisms for reducing the oligomeric size by anionic and cationic detergents with identical apolar parts stresses the importance of charge interactions. Our findings support some aspects of the micelle model of α-crystallin and can be related to its chaperone activity. Accepted: 18 October 1996     

Titrating Theileria parva: single stocks against combination of stocks

Theileria parva is the causative agent of East Coast fever (ECF), an important cattle disease in East and Central Africa. One of the methods for control of ECF is 'infection and treatment', a procedure in which an animal is infected with the live parasite and at the same time treated with a long-acting oxytetracycline formulation, restraining the infection and allowing a protective cellular immune response to develop. Optimal immunizing doses were estimated using models of trichotomous response: dysimmunization (death or severe reaction during immunization), immunization failure (death or severe reaction during lethal challenge) and successful immunization (neither dysimmunization nor immunization failure). In this paper we present methods of interpreting immunization trials and apply these methods to previously unpublished data from two such trials: one with a mixture of three T. parva stocks and one with a single T. parva stock. We explain why titration trials conducted with a cocktail of antigens could predict a suboptimal immunization dose. Indeed it is possible for a combination of three individually efficient stocks to result in a mixture with which optimal immunization response might be difficult to achieve, because of averaging effects. The corresponding interpretation provides insights into why standard immunization trials for T. parva have not yielded the results that might be expected of them. The results of this work may also have implications for the use of antigen cocktails in cancer, HIV and malaria vaccine trials.     

Effects of snout dimensions on the hydrodynamics of suction feeding in juvenile and adult seahorses

Seahorses give birth to juveniles having a fully functional feeding apparatus, and juvenile feeding behaviour shows striking similarities to that of adults. However, a significant allometric growth of the snout is observed during which the snout shape changes from relatively short and broad in juveniles to relatively long and slender in adults. Since the shape of the buccal cavity is a critical determinant of the suction performance, this snout allometry will inevitably affect the suction feeding ability. To test whether the snout is optimised for suction feeding throughout an ontogeny, we simulated the expansion of different snout shapes varying from extremely long and slender to short and broad for juvenile and adult snout sizes, using computational fluid dynamic models. Our results showed that the snout diameter at the start of the simulations is involved in a trade-off between the realizable suction volume and expansion time on the one hand (improving with larger initial diameters), and maximal flow velocity on the other hand (improving with smaller initial diameters). Moreover suction performance (suction volume as well as maximal attainable flow velocity) increased with decreasing snout length. However, an increase in snout length decreases the time to reach the prey by the cranial rotation, which may explain the prevalence of long snouts among syngnathid fishes despite the reduced suction performance. Thus, the design of the seahorse snout revolves around a trade-off between the ability to generate high-volume suction versus minimisation of the time needed to reach the prey by the cranial rotation.     

Potential macro-detritivore range expansion into the subarctic stimulates litter decomposition: a new positive feedback mechanism to climate change?

As a result of low decomposition rates, high-latitude ecosystems store large amounts of carbon. Litter decomposition in these ecosystems is constrained by harsh abiotic conditions, but also by the absence of macro-detritivores. We have studied the potential effects of their climate change-driven northward range expansion on the decomposition of two contrasting subarctic litter types. Litter of Alnus incana and Betula pubescens was incubated in microcosms together with monocultures and all possible combinations of three functionally different macro-detritivores (the earthworm Lumbricus rubellus, isopod Oniscus asellus, and millipede Julus scandinavius). Our results show that these macro-detritivores stimulated decomposition, especially of the high-quality A. incana litter and that the macro-detritivores tested differed in their decomposition-stimulating effects, with earthworms having the largest influence. Decomposition processes increased with increasing number of macro-detritivore species, and positive net diveristy effects occurred in several macro-detritivore treatments. However, after correction for macro-detritivore biomass, all interspecific differences in macro-detritivore effects, as well as the positive effects of species number on subarctic litter decomposition disappeared. The net diversity effects also appeared to be driven by variation in biomass, with a possible exception of net diversity effects in mass loss. Based on these results, we conclude that the expected climate change-induced range expansion of macro-detritivores into subarctic regions is likely to result in accelerated decomposition rates. Our results also indicate that the magnitude of macro-detritivore effects on subarctic decomposition will mainly depend on macro-detritivore biomass, rather than on macro-detritivore species number or identity.     

Identification of the non-lysosomal glucosylceramidase as beta-glucosidase 2

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.     

Analysis and quantification of diagnostic serum markers and protein signatures for Gaucher disease

Novel approaches for the qualitative and quantitative proteomics analysis by nanoscale LC-MS applied to the study of protein expression response in depleted and undepleted serum of Gaucher patients undergoing enzyme replacement therapy are presented. Particular emphasis is given to the method reproducibility of these LC-MS experiments without the use of isotopic labels. The level of chitotriosidase, an established Gaucher biomarker, was assessed by means of an absolute concentration determination technique for alternate scanning LC-MS generated data. Disease associated proteins, including fibrinogens, complement cascade proteins, and members of the high density lipoprotein serum content, were recognized by various clustering methods and sorting and intensity profile grouping of identified peptides. Condition-unique LC-MS protein signatures could be generated utilizing the measured serum protein concentrations and are presented for all investigated conditions. The clustering results of the study were also used as input for gene ontology searches to determine the correlation between the molecular functions of the identified peptides and proteins.     

Neuromechanics: an integrative approach for understanding motor control

Neuromechanics seeks to understand how muscles, sense organs,motor pattern generators, and brain interact to produce coordinatedmovement, not only in complex terrain but also when confrontedwith unexpected perturbations. Applications of neuromechanicsinclude ameliorating human health problems (including prosthesisdesign and restoration of movement following brain or spinalcord injury), as well as the design, actuation and control ofmobile robots. In animals, coordinated movement emerges fromthe interplay among descending output from the central nervoussystem, sensory input from body and environment, muscle dynamics,and the emergent dynamics of the whole animal. The inevitablecoupling between neural information processing and the emergentmechanical behavior of animals is a central theme of neuromechanics.Fundamentally, motor control involves a series of transformationsof information, from brain and spinal cord to muscles to body,and back to brain. The control problem revolves around the specifictransfer functions that describe each transformation. The transferfunctions depend on the rules of organization and operationthat determine the dynamic behavior of each subsystem (i.e.,central processing, force generation, emergent dynamics, andsensory processing). In this review, we (1) consider the contributionsof muscles, (2) sensory processing, and (3) central networksto motor control, (4) provide examples to illustrate the interplayamong brain, muscles, sense organs and the environment in thecontrol of movement, and (5) describe advances in both roboticsand neuromechanics that have emerged from application of biologicalprinciples in robotic design. Taken together, these studiesdemonstrate that (1) intrinsic properties of muscle contributeto dynamic stability and control of movement, particularly immediatelyafter perturbations; (2) proprioceptive feedback reinforcesthese intrinsic self-stabilizing properties of muscle; (3) controlsystems must contend with inevitable time delays that can simplifyor complicate control; and (4) like most animals under a varietyof circumstances, some robots use a trial and error processto tune central feedforward control to emergent body dynamics.     

Development and practical applications of a method for repeated transvaginal, ultrasound-guided biopsy collection of the bovine ovary

In response to the increasing research into primordial and preantral follicular dynamics, a device for transvaginal, ultrasound-guided biopsy collection of the bovine ovary was developed and tested. The new device is based upon a commercially available Ovum Pick-up instrument and consists of a modified needle guidance system, which has been equipped with a trocar needle and caries a 60 cm long true-cut biopsy needle. Biopsies are captured in a 20mm long and 2mm wide specimen notch. In the present experiment, 10 cows were subjected to a twice weekly biopsy regime over a four-week period. A total of 208 attempts at biopsy collection were made, and 141 tissue samples collected (success rate of 68%). Through histological and immunological analyses, these tissue samples have been shown to contain primordial and preantral follicles. At the end of the trial period, several of the donor cows were slaughtered at timed intervals, and the ovaries were harvested for assessment of the damage inflicted by the repeated biopsy procedure. Post mortem ovaries were inspected macroscopically and examined by conventional histological staining. In ovaries retrieved 2 days after the last biopsy session, blood clots were macroscopically apparent throughout the ovaries. Histological examination showed increased infiltration of red blood cells in the ovarian stroma. Analysis from ovaries collected at subsequent slaughter points revealed reduced infiltration of blood, and clear indications of resumed antral follicle development were apparent towards the end of the first month after the trial period. We conclude that the biopsy sampling technique is a repeatable procedure which could serve as a renewable source of primordial and preantral follicles for culture, and as an in vitro model for the study of preantral follicular dynamics.     

Ferrous Iron Stimulates Phenol Oxidase Activity and Organic Matter Decomposition in Waterlogged Wetlands

Soil organic matter decomposition is limited at waterlogged conditions by the low activity of extracellular enzymes like phenol oxidases. In this paper, we show that ferrous iron (Fe²⁺), which is abundant in waterlogged soils, significantly stimulates phenol oxidase activity both in pure enzyme assays and in waterlogged soil slurries from nutrient-poor dune slacks. However, the effects in soil slurries were less strong than in enzyme assays. Both the addition of Fe²⁺ and the initial presence of Fe²⁺ stimulated phenol oxidase activity at the microaerophilic conditions tested. This stimulation is attributed to the catalysis of additional OH radical production, promoting the oxidation of phenolics. Subsequently, the presence of Fe²⁺ strongly increased total decomposition rates of soil organic matter, measured as CO₂ production and Cotton strip Tensile Strength Loss. There is circumstantial evidence that this stimulation by Fe²⁺ could be important for decomposition in wetlands at field conditions, but its relevance compared to the effects of other compounds still needs to be elucidated. These results emphasise the crucial role of water quality in determining extracellular enzyme activity and decomposition in waterlogged wetlands.