Inactivation of glyceraldehyde 3-phosphate dehydrogenase by sugars, prednisolone-21-hemisuccinate, cyanate and other small molecules

Diabetes, diarrhoea, renal failure and glucocorticoid therapy have all been identified as independent risk factors for cataract. Increased post-translational modification of proteins, leading to inactivation of enzymes and induction of conformational changes within proteins could result in lens opacification and cataract. Aspirin has been associated with many beneficial effects, including protection against cataract, in-vivo. alpha-Crystallin has been shown to act as a molecular chaperone in-vitro. This lenticular protein prevented the thermal aggregation of other lens proteins in-vitro and has sequence and functional homology with the small heat shock proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) is constitutively expressed in tissues and is susceptible to chemical modification in-vivo. In-vitro incubations of GAP-DH with sugars, cyanate and prednisolone-21-hemisuccinate, all led to significant loss of enzyme activity with time in two buffer systems. Rapid inactivation occurred when GAP-DH was incubated with fructose 6-phosphate or prednisolone-21-hemisuccinate. Slower inactivation was observed when GAP-DH was incubated with fructose, glucose 6-phosphate or potassium cyanate. Glucose did not inactivate GAP-DH under the conditions of our experiments. Aspirin and ibuprofen were shown to inactivate GAP-DH very rapidly in-vitro. Bovine lenticular alpha-crystallin conferred no protection against GAP-DH inactivation. This is the first occasion that alpha-crystallin has been demonstrated to be unable to protect against inactivation in our chemical enzyme inactivation system. This may have implications for the susceptibility of lenticular GAP-DH to post-translational inactivation.     

Subunit composition of neurofilaments specifies axonal diameter

Neurofilaments (NFs), which are composed of NF-L, NF-M, and NF-H, are required for the development of normal axonal caliber, a property that in turn is a critical determinant of axonal conduction velocity. To investigate how each subunit contributes to the radial growth of axons, we used transgenic mice to alter the subunit composition of NFs. Increasing each NF subunit individually inhibits radial axonal growth, while increasing both NF-M and NF-H reduces growth even more severely. An increase in NF-L results in an increased filament number but reduced interfilament distance. Conversely, increasing NF-M, NF-H, or both reduces filament number, but does not alter nearest neighbor interfilament distance. Only a combined increase of NF-L with either NF-M or NF-H promotes radial axonal growth. These results demonstrate that both NF-M and NF-H play complementary roles with NF-L in determining normal axonal calibers.     

Steering without representation with the use of active fixation

This paper demonstrates the use of active fixation on both fixed and moving fixation points to guide a robot vehicle by means of a steering rule which, at large distances, sets the steering angle directly proportional to the deviation of gaze direction from translation direction. Steering a motor vehicle around a winding but otherwise uncluttered road has been observed by Land and Lee to involve repeated periods of visual fixation upon the tangent point of the inside of each bend. We suggest that proportional rule devised for steering in the robotic example appears applicable to the observed human performance data, providing an alternative explanation to the quadratic rule proposed by Land and Lee.     

Encapsulated islet cells: the role of direct and indirect presentation and the relevance to xenotransplantation and autoimmune recurrence

Immuno-isolation of islet tissue for the purpose of curing diabetes without immunosuppression is an attractive concept that has been extensively investigated over the last 20 years. Rather than give a detailed review of the past and present history of the immuno-isolation or encapsulation approach for islet transplantation, this paper will examine the concepts behind encapsulation, concentrating on the immune mechanisms involved.     

Role of the propeptide and gamma-glutamic acid domain of factor IX for in vitro carboxylation by the vitamin K-dependent carboxylase

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the processive carboxylation of specific glutamates in a number of proteins related to blood coagulation and bone. To address the independent importance of the propeptide, gamma-carboxyglutamic acid (Gla) domain and elements beyond the Gla domain of factor IX in vitamin K-dependent carboxylation, we have examined the kinetics of carboxylation of peptides containing (1) propeptide and Gla domain, (2) the Gla domain alone, (3) uncarboxylated bone Gla protein, (4) propeptide followed by the entire uncarboxylated factor IX molecule, and (5) the factor IX propeptide followed by a non-Gla domain sequence. Our studies indicate that peptides with a covalently linked propeptide have Km values similar to the physiological substrate of the carboxylase. In contrast, the Gla domain of factor IX has a >/=230-fold higher Km for the carboxylase than the corresponding peptide with a covalently linked propeptide. This contrasts with bone Gla protein, another vitamin K-dependent protein, which appears not to require a covalently linked propeptide for high-affinity binding to the carboxylase. Analysis of the carboxylation products of a propeptide/non-Gla domain substrate indicate that it is carboxylated multiple times in a processive manner. These studies show that the perceived binding affinity of the carboxylase substrate and processivity is conferred by the propeptide without requiring the conserved Gla domain sequences and that factor IX and bone Gla protein may have distinct mechanisms of interacting with the carboxylase.     

Sensitive fused-silica capillary gas chromatographic assay using electron-capture detection for indomethacin in ovine fetal fluids

A sensitive gas chromatographic (GC) method with electron-capture detection (ECD) has been developed to quantitate indomethacin (IND) in plasma, urine, amniotic, and tracheal fluids obtained from the pregnant sheep model. IND and the internal standard, alpha-methylindomethacin (alpha-Me-IND) are extracted by a simple liquid-liquid extraction procedure using ethyl acetate and derivatized with N-methyl-N-(tert.-butyldimethyl-silyl)trifluoroacetamide (MTBSTFA) at 60 degrees C for 50 min. The limit of quantitation (LOQ) is 1 ng/ml with a C.V. < 10% and signal-to-noise ratio > 10. Recoveries from all fluids were greater than 80%. Calibration curves were linear over the range of 1-32 ng/ml with a coefficient of determination (r2) > 0.999. Inter- and intra-day coefficients of variation were < 10% at concentrations of 2-32 ng/ml, and < 20% at the LOQ. Applicability of the developed method is demonstrated for a pharmacokinetic study of IND samples collected following long-term infusion of IND in a chronically instrumented ovine fetus.