Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels.

Early changes in biochemical markers of bone formation during teriparatide therapy correlate with improvements in vertebral strength in men with glucocorticoid-induced osteoporosis

Summary Changes of the bone formation marker PINP correlated positively with improvements in vertebral strength in men with glucocorticoid-induced osteoporosis (GIO) who received 18-month treatment with teriparatide, but not with risedronate. These results support the use of PINP as a surrogate marker of bone strength in GIO patients treated with teriparatide. Introduction To investigate the correlations between biochemical markers of bone turnover and vertebral strength estimated by finite element analysis (FEA) in men with GIO. Methods A total of 92 men with GIO were included in an 18-month, randomized, open-label trial of teriparatide (20 μg/day, n = 45) and risedronate (35 mg/week, n = 47). High-resolution quantitative computed tomography images of the 12th thoracic vertebra obtained at baseline, 6 and 18 months were converted into digital nonlinear FE models and subjected to anterior bending, axial compression and torsion. Stiffness and strength were computed for each model and loading mode. Serum biochemical markers of bone formation (amino-terminal-propeptide of type I collagen [PINP]) and bone resorption (type I collagen cross-linked C-telopeptide degradation fragments [CTx]) were measured at baseline, 3 months, 6 months and 18 months. A mixed-model of repeated measures analysed changes from baseline and between-group differences. Spearman correlations assessed the relationship between changes from baseline of bone markers with FEA variables. Results PINP and CTx levels increased in the teriparatide group and decreased in the risedronate group. FEA-derived parameters increased in both groups, but were significantly higher at 18 months in the teriparatide group. Significant positive correlations were found between changes from baseline of PINP at 3, 6 and 18 months with changes in FE strength in the teriparatide-treated group, but not in the risedronate group. Conclusions Positive correlations between changes in a biochemical marker of bone formation and improvement of biomechanical properties support the use of PINP as a surrogate marker of bone strength in teriparatide-treated GIO patients.

The “Out of Africa Tribe” (II): Paleolithic warriors with big canoes and protective weapons

It is generally difficult to establish a timeline for the appearance of different technologies and tools during human cultural evolution. Here I use stochastic character mapping of discrete traits using human mtDNA phylogenies rooted to the Reconstructed Sapiens Reference Sequence (RSRS) as a model to address this question. The analysis reveals that the ancestral state of Homo sapiens was hunting, using material innovations that included bows and arrows, stone axes and spears. However, around 80,000 y before present, a transition occurred, from this ancestral hunting tradition, toward the invention of protective weapons such as shields, the appearance of ritual fighting as a socially accepted behavior and the construction of war canoes for the fast transport of large numbers of warriors. This model suggests a major cultural change, during the Palaeolithic, from hunters to warriors. Moreover, in the light of the recent Out of Africa Theory, it suggests that the “Out of Africa Tribe” was a tribe of warriors that had developed protective weapons such as shields and used big war canoes to travel the sea coast and big rivers in raiding expeditions.

Extended kalman filter for estimation of parameters in nonlinear state-space models of biochemical networks.

It is system dynamics that determines the function of cells, tissues and organisms. To develop mathematical models and estimate their parameters are an essential issue for studying dynamic behaviors of biological systems which include metabolic networks, genetic regulatory networks and signal transduction pathways, under perturbation of external stimuli. In general, biological dynamic systems are partially observed. Therefore, a natural way to model dynamic biological systems is to employ nonlinear state-space equations. Although statistical methods for parameter estimation of linear models in biological dynamic systems have been developed intensively in the recent years, the estimation of both states and parameters of nonlinear dynamic systems remains a challenging task. In this report, we apply extended Kalman Filter (EKF) to the estimation of both states and parameters of nonlinear state-space models. To evaluate the performance of the EKF for parameter estimation, we apply the EKF to a simulation dataset and two real datasets: JAK-STAT signal transduction pathway and Ras/Raf/MEK/ERK signaling transduction pathways datasets. The preliminary results show that EKF can accurately estimate the parameters and predict states in nonlinear state-space equations for modeling dynamic biochemical networks.

Bioavailability of silver nanoparticles and ions: from a chemical and biochemical perspective

Owing to their antimicrobial properties, silver nanoparticles (NPs) are the most commonly used engineered nanomaterial for use in a wide array of consumer and medical applications. Many discussions are currently ongoing as to whether or not exposure of silver NPs to the ecosystem (i.e. plants and animals) may be conceived as harmful or not. Metallic silver, if released into the environment, can undergo chemical and biochemical conversion which strongly influence its availability towards any biological system. During this process, in the presence of moisture, silver can be oxidized resulting in the release of silver ions. To date, it is still debatable as to whether any biological impact of nanosized silver is relative to either its size, or to its ionic constitution. The aim of this review therefore is to provide a comprehensive, interdisciplinary overview--for biologists, chemists, toxicologists as well as physicists--regarding the production of silver NPs, its (as well as in their ionic form) chemical and biochemical behaviours towards/within a multitude of relative and realistic biological environments and also how such interactions may be correlated across a plethora of different biological organisms.

Molecular and biochemical analysis of the {\it Drosophila\/} segmentation gene {\it runt\/}

Genetic evidence has indicated that the segmentation gene runt plays a key role in regulating gene expression of the pair-rule genes hairy, even-skipped, and fushi tarazu. In contrast to other pair-rule genes, sequence data of the runt open reading frame did not reveal homologies to DNA-binding motifs of known transcriptional regulatory proteins. This thesis project examined several properties of the runt gene based on the sequence of the transcription unit, including the subcellular localization of the protein in vivo, its ability to bind DNA, and the functionality of a putative nucleotide binding domain.^ A runt-specific antibody was generated and used to demonstrate that runt is localized in the nucleus. Since the precise overlap of the pair-rule stripes is thought to be critical for the determination of cellular identity along the anterior-posterior axis, phasing of early runt expression in the blastoderm was examined with regard to the segmentation genes hairy, even-skipped, and fushi tarazu. runt was also expressed at later stages of embryogenesis, including expression in neuroblasts, and ganglion mother cells of the developing nervous system. Expression at this stage was required for the subsequent formation of specific neurons and runt was extensively expressed in the central and peripheral nervous systems.^ Several experiments were done to address the biochemical function of the runt protein. A direct interaction of runt with DNA was first examined. Although bacterial expressed runt was found to bind dsDNA-cellulose, subsequent experiments failed to detect sequence-specific interactions with DNA. Inter-species conservation of the putative nucleotide binding domain suggested that this region was functionally important, and runt protein bound a labeled ATP analog with high affinity in vitro. Finally, the effect of substitution of a critical residue of the nucleotide binding domain on runt activity was examined in vivo. Ectopic expression of the mutant protein indicated that this conserved substitution altered, but did not eliminate, runt activity as evaluated by segmentation phenotype and viability. ^

Biochemical characterization of PICK1, a PKC binding protein

Protein kinase C (PKC) is a family of serine-threonine kinases that are activated by a wide variety of hormones, neurotransmitters and growth factors. A single cell type contains multiple isoforms that are translocated to distinct and different subcellular sites upon mitogenic stimulus. Many different cellular responses are attributed to PKC activity though relatively few substrates or binding proteins have been definitively characterized. We used the hinge and catalytic domain of PKC$\alpha$ (PKC7) in a yeast two-hybrid screen to clone proteins that interact with C-kinase (PICKs). One protein which we have termed PICK1 may be involved in PKC$\alpha$-specific function at the level of the nuclear membrane after activation. Binding of PICK1 to PKC$\alpha$ has been shown to be isoform specific as it does not bind to PKC$\beta$II or PKC$\alpha$ in the yeast two-hybrid system. PICK1 mRNA expression level is highest in testis and brain with lower levels of expression in skeletal muscle, heart, kidney, lung and liver. PICK1 protein contains five PKC consensus phosphorylation sites and serves as an in vitro substrate for PKC. The PICK1 protein also contains a P-Loop motif that has been shown to bind ATP or GTP in the Ras family of oncoproteins as well as the G-Protein family. Proteins which bind ATP or GTP using this motif all have some sort of catalytic function although none has been identified for PICK1 as yet. PICK1 contains a DHR/GLGF motif at the N-terminus of the protein. The DHR/GLGF motif is contained in a number of recently described proteins and has been shown to mediate protein-protein interactions at the level of membranes and cytoskeleton. When both PKC$\alpha$ and PICK1 are co-expressed in Cos1 cells the two proteins co-localize to the perinucleus in immunoflouresence studies and co-immunoprecipitate. The binding site for PKC7 has been localized to amino acids 1-358 on PICK1 which contains the DHR/GLGF motif. Binding of PICK1 to PKC$\alpha$ requires the hinge and C-terminal domains of PKC$\alpha$. In vitro, PICK1 binds to PKC$\alpha$ and inhibits its activity as assayed by myelin basic protein phosphorylation. PICK1 also binds to TIS21, a primary response gene that is expressed in response to phorbol ester and growth factor treatment. The Caenorhabditis elegans homologue of PICK1 has been cloned and sequenced revealing a high degree of conservation in the DHR/GLGF motif. A more C-terminal region also shows a high degree of conservation, and the C. elegans PICK1 homologue binds to PKC7 suggesting a conservation of function. Taken together these results suggest that PICK1 may be involved in a PKC$\alpha$-specific function at the level of the nuclear membrane. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

BIOCHEMICAL DETERMINANTS IN THE CYTOTOXIC ACTION OF 9-BETA- D- ARABINOFURANOSYLADENINE

Studies with 9-(beta)-D-arabinfuranosyladenine (ara-A) have illustrated that there are numerous cell functions which are affected by the nucleoside analog and its metabolites. Although the precise mechanism responsible for the cytotoxicity of this drug is not known, it is presently thought tha