Factor XSanto Domingo. Evidence that the severe clinical phenotype arises from a mutation blocking secretion.

Factor X (FX) is a vitamin K-dependent plasma protein required for the intrinsic and extrinsic pathways of blood coagulation. FXSanto Domingo is a hereditary FX deficiency which is characterized clinically by a severe bleeding diathesis. The proposita has a FX activity of less than 1% and a FX antigen of less than 5%. We have determined the molecular basis of the defect in the FXSanto Domingo gene by amplification of all eight exons with polymerase chain reaction and subsequent sequence analysis. The patient is homozygous for a G----A transition in exon I at codon -20 (numbering the alanine at the NH2 terminus of the mature protein as +1), resulting in the substitution of arginine for glycine in the carboxy-terminal part of the signal peptide. This amino acid change occurs near the presumed cleavage site of the signal peptidase. We hypothesized that the mutation might prevent cleavage by the signal peptidase which in turn would impair proper secretion of the FX protein. To test this hypothesis, we compared the expression of wild type and mutant FX cDNA in a human kidney cell line. Wild type and mutant constructs in the expression vector pCMV4 were introduced into the human embryonic kidney cell line 293 by calcium phosphate transfection. FX antigen levels in the supernatant of the cells harboring the wild type construct were 2.4 micrograms/10(7) cells per 24 h, whereas antigen levels in media from cells containing the FXSanto Domingo construct were undetectable. No FX antigen was detected in the cell lysates of cells transfected with the mutant construct. To insure that the difference in protein levels was not due to a difference in steady state levels of mRNA, Northern analysis was performed on RNA from the cell lysates of both constructs. The results showed a transcript of the same size, present in roughly equal amounts, in both cases. Thus, the defect in the signal sequence of FXSanto Domingo exerts its effect posttranscriptionally. FXSanto Domingo is the first described example of a bleeding diathesis due to a mutation in the signal sequence

Lipase-catalyzed Reactions at Interfaces of Two-phase Systems and Microemulsions

This work describes the influence of two polar lipids, Sn-1/3 and Sn-2 monopalmitin, on the activity of lipase in biphasic systems and in microemulsions. In previous communications, we have shown that Sn-2 monoglycerides can replace Sn-1,3 regiospecific lipases at the oil–water interface, causing a drastically reduced rate of lipolysis. We here demonstrate that even if the lipase is expelled from the interface, it can catalyze esterification of the Sn-2 monoglyceride with fatty acids in both macroscopic oil–water systems and in microemulsions, leading to formation of di- and triglycerides

Planning and problem-solving training for patients with schizophrenia: a randomized controlled trial

BACKGROUND: The purpose of this study was to assess whether planning and problem-solving training is more effective in improving functional capacity in patients with schizophrenia than a training program addressing basic cognitive functions. METHODS: Eighty-nine patients with schizophrenia were randomly assigned either to a computer assisted training of planning and problem-solving or a training of basic cognition. Outcome variables included planning and problem-solving ability as well as functional capacity, which represents a proxy measure for functional outcome. RESULTS: Planning and problem-solving training improved one measure of planning and problem-solving more strongly than basic cognition training, while two other measures of planning did not show a differential effect. Participants in both groups improved over time in functional capacity. There was no differential effect of the interventions on functional capacity. CONCLUSION: A differential effect of targeting specific cognitive functions on functional capacity could not be established. Small differences on cognitive outcome variables indicate a potential for differential effects. This will have to be addressed in further research including longer treatment programs and other settings

Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity

Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher’s disease. More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson’s disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (∼99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging

The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport

The steady-state and pre-steady-state kinetics of glutamate transport by the neuronal glutamate transporter EAAC1 were determined under conditions of outward glutamate transport and compared to those found for the inward transport mode. In both transport modes, the glutamate-induced current is composed of two components, the coupled transport current and the uncoupled anion current, and inhibited by a specific non-transportable inhibitor. Furthermore, the glutamate-independent leak current is observed in both transport modes. Upon a glutamate concentration jump outward transport currents show a distinct transient phase that deactivates within 15 ms. The results demonstrate that the general properties of EAAC1 are symmetric, but the rates of substrate transport and anion flux are asymmetric with respect to the orientation of the substrate binding site in the membrane. Therefore, the EAAC1 anion conductance differs from normal ligand-gated ion channels in that it can be activated by glutamate and Na+ from both sides of the membrane

Is the glutamate residue Glu-373 the proton acceptor of the excitatory amino acid carrier 1?

Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na+/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na+binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na+/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes

Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds

The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft, and thus, is crucial for proper excitatory signaling. Initial steps in the process of glutamate transport take place within a time scale of microseconds to milliseconds. Here we compare the steady-state and pre-steady-state kinetics of the neuronal heterologously expressed glutamate transporter EAAC1, cloned from the mammalian retina. Rapid transporter dynamics, as measured by using whole-cell current recordings, were resolved by applying the laser-pulse photolysis technique of caged glutamate with a time resolution of 100 μs. EAAC1-mediated pre-steady-state currents are composed of two components: A transport current generated by substrate-coupled charge translocation across the membrane and an anion current that is not stoichiometrically coupled to glutamate transport. The two currents were temporally resolved and studied independently. Our results indicate a rapid glutamate-binding step occurring on a submillisecond time scale that precedes subsequent slower electrogenic glutamate translocation across the membrane within a few milliseconds. The voltage-dependent steady-state turnover time constant of the transporter is about 1/10 as fast, indicating that glutamate translocation is not rate limiting. A third process, the transition to an anion-conducting state, is delayed with respect to the onset of glutamate transport. These rapid transporter reaction steps are summarized in a sequential shuttle model that quantitatively accounts for the results obtained here and are discussed regarding their functional importance for glutamatergic neurotransmission in the central nervous system