PLASMA PHENYLALANINE DETERMINATION BY QUANTITATIVE DENSITOMETRY OF THIN LAYER CHROMATOGRAMS AND BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY IN RELATION WITH MODERN MANAGEMENT OF PHENYLKETONURIA.

BACKGROUND: The modern management of phenylketonuria (PKU) consists of generalized newborn screening (NBS) for hyperphenylalaninemia (HPA), confirmation of HPA in children detected in the NBS, introduction of dietary treatment in the first weeks of life, followed by monitoring the treatment of PKU for decades to maintain phenylalaninemia within the limits that will not affect the brain. The present study aimed to evaluate the usefulness of two chromatographic methodologies for determination of plasma Phe level in the routine management of PKU: the two dimensional thin layer chromatography (2D - TLC) and the high performance liquid chromatography (HPLC) procedures, respectively. MATERIAL AND METHODS: Samples of blood from 23 children with HPA detected by neonatal screening or with confirmed PKU who received treatment by low-Phe diet were analyzed to estimate the plasma Phe level by the two chromatographic procedures. RESULTS: In case of three subjects the very low concentrations of plasma Phe could not be detected by the 2D - TLC methodology, since the spot was not visible on the chromatogram. In four patients the differences between the values of plasma Phe determined by the two methodologies are not statistically significant, while in fifteen subjects the differences are highly statistically significant. This is due to the greater errors that appear in the case of 2D - TLC methodology. In the range of concentrations of plasma Phe higher than 360 µmol/L (which is the cut-off value for HPA), although in four cases there were statistically significant differences in the level of plasma Phe determined by the two methodologies, the value obtained by the 2D - TLC methodology was high enough to influence the decision of changing the diet so that HPA is kept under control. In addition, the intense spot of Phe on the 2D - TLC chromatogram may be detected even by un unexperienced laboratory specialist. CONCLUSION: The HPLC procedure for measurement of plasma Phe level is very suitable to be used in the routine management of PKU. The 2D - TLC procedure may be accompanied by relatively high errors; however, it detects patients with severe PKU.

COMPARISON OF PLASMA PHENYLALANINE DETERMINATION BY DENSITOMETRY OF THIN-LAYER CHROMATOGRAMS AND BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY IN RELATION WITH THE SCREENING OF PHENYLKETONURIA.

OBJECTIVE: To compare two chromatographic methodologies for determination of plasma phenylalanine (Phe) and their usefulness for diagnosing hyperphenylalaninemia (HPA) and phenylketonuria (PKU). METHODS: The plasma amino acids were isolated and concentrated from blood collected from infants with HPA detected by newborn screening. The plasma Phe was determined in parallel by HPLC and by image-densitometry of 2D-TLC plates. RESULTS: Typical examples of 2D-TLC plates and HPLC chromatograms from infants with HPA and PKU are presented and evaluated. The Phe spot was visible on 2D - TLC plates at Phe concentrations higher than 300 µmol/L. The standard calibration curve traced after image-densitometry of the Phe spot presented high dispersion of values at each concentration of Phe, high SD values, the equation of the curve having a low R-squared value (0.862). In contrast, the standard calibration curve obtained by HPLC shows linearity on the range of concentrations from 100 - 16,000 µmol/L, extremely small SD values, the equation of the curve has a very high R-squared value (0.999). CONCLUSIONS: The HPLC methodology is appropriate to confirm HPA detected by newborn or selective screening of PKU. The 2D - TLC methodology is adequate to detect patients with severe PKU.

Recent progress in elucidating signalling proteolytic pathways in muscle wasting: potential clinical implications.

AIMS: Muscle wasting prevails with disuse (bedrest and immobilisation) and is associated with many diseases (cancer, sepsis, diabetes, kidney failure, trauma, etc.). This results first in prolonged hospitalisation with associated high health-care costs and second and ultimately in increased morbidity and mortality. The precise characterisation of the signalling pathways leading to muscle atrophy is therefore particularly relevant in clinical settings. DATA SYNTHESIS: Recent major papers have identified highly complex intricate pathways of signalling molecules, which induce the transcription of the muscle-specific ubiquitin protein ligases MAFbx/Atrogin-1 and MuRF1 that are overexpressed in nearly all muscle wasting diseases. These signalling pathways have been targeted with success in animal models of muscle wasting. In particular, these findings have revealed a finely tuned crosstalk between both anabolic and catabolic processes. CONCLUSIONS: Whether or not such strategies may be useful for blocking or at least limiting muscle wasting in weight losing and cachectic patients is becoming nowadays a very exciting clinical challenge.

In vitro radiosensitizing effects of ultrasmall gadolinium based particles on tumour cells.

Since radiotherapy is widely used in cancer treatment, it is essential to develop strategies which lower the irradiation burden while increasing efficacy and become efficient even in radio resistant tumors. Our new strategy is relying on the development of solid hybrid nanoparticles based on rare-earth such as gadolinium. In this paper, we then evidenced that gadolinium-based particles can be designed to enter efficiently into the human glioblastoma cell line U87 in quantities that can be tuned by modifying the incubation conditions. These sub-5 nm particles consist in a core of gadolinium oxide, a shell of polysiloxane and are functionalized by diethylenetriaminepentaacetic acid (DTPA). Although photoelectric effect is maximal in the [10-100 keV] range, such particles were found to possess efficient in-vitro radiosensitizing properties at an energy of 660 keV by using the "single-cell gel electrophoresis comet assay," an assay that measures the number of DNA damage that occurs during irradiation. Even more interesting, the particles have been evidenced by MTT assays to be also efficient radiosensitizers at an energy of 6 MeV for doses comprised between 2 and 8 Gy. The properties of the gadolinium-based particles give promising opening to a particle-assisted radio-therapy by using irradiation systems already installed in the majority of hospitals.

Phenomenological modeling of tumor diameter growth based on a mixed effects model.

Over the last few years, taking advantage of the linear kinetics of the tumor growth during the steady-state phase, tumor diameter-based rather than tumor volume-based models have been developed for the phenomenological modeling of tumor growth. In this study, we propose a new tumor diameter growth model characterizing early, late and steady-state treatment effects. Model parameters consist of growth rhythms, growth delays and time constants and are meaningful for biologists. Biological experiments provide in vivo longitudinal data. The latter are analyzed using a mixed effects model based on the new diameter growth function, to take into account inter-mouse variability and treatment factors. The relevance of the tumor growth mixed model is firstly assessed by analyzing the effects of three therapeutic strategies for cancer treatment (radiotherapy, concomitant radiochemotherapy and photodynamic therapy) administered on mice. Then, effects of the radiochemotherapy treatment duration are estimated within the mixed model. The results highlight the model suitability for analyzing therapeutic efficiency, comparing treatment responses and optimizing, when used in combination with optimal experiment design, anti-cancer treatment modalities.

Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats.

Little information is available on proteolytic pathways responsible for muscle wasting in cancer cachexia. Experiments were carried out in young rats to demonstrate whether a small (< 0.3% body weight) tumor may activate the lysosomal, Ca(2+)-dependent, and/or ATP-ubiquitin-dependent proteolytic pathway(s) in skeletal muscle. Five days after tumor implantation, protein mass of extensor digitorum longus and tibialis anterior muscles close to a Yoshida sarcoma was significantly reduced compared to the contralateral muscles. According to in vitro measurements, protein loss totally resulted from increased proteolysis and not from depressed protein synthesis. Inhibitors of lysosomal and Ca(2+)-dependent proteases did not attenuate increased rates of proteolysis in the atrophying extensor digitorum longus. Accordingly, cathepsin B and B+L activities, and mRNA levels for cathepsin B were unchanged. By contrast, ATP depletion almost totally suppressed the increased protein breakdown. Furthermore, mRNA levels for ubiquitin, 14 kDa ubiquitin carrier protein E2, and the C8 or C9 proteasome subunits increased in the atrophying muscles. Similar adaptations occurred in the muscles from cachectic animals 12 days after tumor implantation. These data strongly suggest that the activation of the ATP-ubiquitin-dependent proteolytic pathway is mainly responsible for muscle atrophy in Yoshida sarcoma-bearing rats.

Nucleotide sequence of bovine preprocathepsin B. A study of polymorphism in the protein coding region.

A cDNA encoding bovine procathepsin B was isolated. The deduced amino acid sequence revealed that a stop (TAG) codon, instead of a Trp-257 codon (TGG), generates in bovine a cathepsin B precursor four amino acids shorter than in other species. Because micro-heterogeneities were previously reported in the cathepsin B primary structure, sequence polymorphism in the protein coding region was then investigated by PCR sequencing of genomic fragments and RNase protection assays. Experiments performed with 12-15 animals of three breeds did not reveal any difference with our cDNA sequence. We conclude that sequence polymorphism in bovine cathepsin B is a rare event, and can only result from the expression of different alleles of a unique gene.

Effect of fasting and thyroidectomy on cysteine proteinase activities in liver and muscle.

Prolonged starvation mimics chronic negative nitrogen balance observed in many physiopathological situations. During starvation, an initial decrease in protein utilization (phase I) is followed by a long period of protein sparing (phase II) that ends with a marked rise in nitrogen excretion (phase III). Variations in protein metabolism during starvation are determined by changes in protein synthesis and degradation rates (Cherel, Y., Attaix, D. Rosolowska-Huszcz, D., Belkhou, R., Robin, J.P., Arnal, M. and Le Maho, Y. (1991) Clin. Sci. 81, 611-619), but little information is available on expression of proteolytic systems. In this study, cathepsin B, H and L activities were compared in hindlimb muscles and liver at various phases of starvation in thyroidectomized and sham-operated rats. In muscle, cathepsin activities fell from the fed state to phase II, which suggests that cathepsins may play a role in the curtailment of muscle proteolysis during protein sparing phase. This decrease of muscle cathepsin activities was reproduced by thyroidectomy alone. In contrast, liver cathepsin B and H activities fell during starvation, but were not affected by thyroidectomy alone. Liver cathepsin L decreased only during starvation in thyroidectomized animals. These observations emphasize that different mechanisms modulate cathepsin expression in skeletal muscle and liver.

A Model-Based Pharmacokinetics Characterization Method of Engineered Nanoparticles for Pilot Studies.

Recent developments on engineered multifunctional nanomaterials have opened new perspectives in oncology. But assessment of both quality and safety in nanomedicine requires new methods for their biological characterization. This paper proposes a new model-based approach for the pre-characterization of multifunctional nanomaterials pharmacokinetics in small scale in vivo studies. Two multifunctional nanoparticules, with and without active targeting, designed for photodynamic therapy guided by magnetic resonance imaging are used to exemplify the presented method. It allows to the experimenter to rapidly test and select the most relevant PK model structure planned to be used in the subsequent explanatory studies. We also show that the model parameters estimated from the in vivo responses provide relevant preliminary information about the tumor uptake, the elimination rate and the residual storage. For some parameters, the accuracy of the estimates is accurate enough to compare and draw significant pre-conclusions. A third advantage of this approach is the possibility to optimally refine the in vivo protocol for the subsequent explanatory and confirmatory studies complying with the 3Rs (reduction, refinement, replacement) ethical recommendations. More precisely, we show that the identified model may be used to select the appropriate duration of the MR imaging sessions planned for the subsequent studies. The proposed methodology integrates MRI image processing, continuous-time system identification algorithms and statistical analysis. Except, the choice of the model parameters to be compared and interpreted, most of the processing procedure may be automated to speed up the PK characterization process at an early stage of experimentation.

Gene structure of mouse cathepsin B.

The structure of a genomic DNA fragment encoding mouse cathepsin B was characterized. The genomic insert spans 15 kbp and contains 9 exons encoding the 339 amino acid residues of mouse preprocathepsin B. Intron break-points are not found at the junctions of the pre-peptide, pro-peptide and mature enzyme. Like other cysteine proteinase genes, the region around the cysteinyl active site is split by an intron, but in contrast with cathepsins L and H the intron break-point is located immediately after the active site.

Multiple leader sequences for mouse cathepsin B mRNA?

We previously described the gene structure of murine cathepsin B. Our results suggested that the 5'-untranslated region (leader) is interrupted by a large intron. The second exon (exon-2) contains the translation initiation site. To characterize the leader region, a rapid amplification of cDNA ends (RACE) procedure was developed. The PCR products were directly cloned and sequenced. Nucleotide sequence analyses revealed three different 5'-cDNA ends, suggesting the existence of three different leader regions. In addition to the leader (LA) previously characterized, we now describe two other 5'-untranslated regions, LB and LC. Leader LB is located 2.3 kb upstream exon-2, and leader LC corresponds to the 3'-end of the first intron and is thus contiguous to exon-2. Our results suggest for murine cathepsin B gene the presence of multiple promoters, and possibly the expression of multiple mRNAs differing in their leader region.

A novel class of inactive steroid-binding sites in female rat liver nuclei.

Nuclear salt extracts from intact female rat liver showed insignificant levels of progesterone-, oestradiol-, testosterone- or dexamethasone-specific binding. However, brief exposure of nuclear extracts to dextran-coated charcoal (DCC) induced binding for all the above classes of steroids. This 'DCC-effect', which was reproduced neither by gel filtration nor by extensive dialysis of the nuclear extract, could not be ascribed to removal of endogenous free or loosely bound steroids. We show that rat liver nuclei contain a class of secondary binding sites (BSII), which exhibit moderate or low affinity for steroid ligands, positive co-operativity, and cross-reaction between classes of steroids. The capacity of BSII sites to bind steroids depends strictly on prior neutralization by DCC of endogenous heat-stable non-dialysable inhibitor(s). The putative roles of these BSII binding sites are discussed in relation to component(s) probably responsible for inhibitory activity.

Species variations amongst proteinases in liver lysosomes.

Cathepsin B, H, L and D activities in liver lysosomes were compared between species. Although cathepsin B and D were detected in bovine, pig, chicken and rat liver, striking species differences were evident for cathepsin H and L. Cathepsin L activity was particularly high in chicken lysosomal extracts, but could not be detected in bovine and pig extracts. Whereas there was no significant cathepsin H activity in bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H activity.

Muscle differentiation in the bovine fetus: a histological and histochemical approach.

The chronology of muscle fiber differentiation was analysed in 37 fetal calves of 69 to 266 days of age. Semitendinosus muscle weight was measured throughout the experimental period and biochemical, histological and histochemical investigations were made to determine respectively the protein and DNA content of the muscle, the size and the number of the fibers and their ATPase and SDH activity. The relative growth of all the quantitative characteristics (muscle weight, protein and DNA content) was much greater in the early stages of gestation than in the new-born animal. In the younger fetuses DNA relative growth was faster than protein relative growth, whereas at the end of gestation the reverse progression was observed. Before 90 days, the muscle tissue was composed of myotube-like cells without any clear organization. The organization of muscle tissue into clear bundles occurred around 120 days of age, and about 30 days later the large myotubes transformed into myofibers. The myotubes reacted positively for acid-ATPase activity, whereas the large population of smaller cells which developed in parallel did not. The number of muscle cells increased up to 240 days of age, as did the percentage of fibers positive for acid-ATPase activity. Finally, oxidative differentiation occurred around 260 days of age, with the appearance of a population of cells characterized by increased SDH activity. A comparison of these results with previous findings suggests that the muscular tissue differentiates through similar stages in various species, but over different lengths of time. The percentage of mature weight might provide a better inter-species time scale than chronological age.

Bimodal collagen fibril diameter distributions direct age-related variations in tendon resilience and resistance to rupture.

Scaling relationships have been formulated to investigate the influence of collagen fibril diameter (D) on age-related variations in the strain energy density of tendon. Transmission electron microscopy was used to quantify D in tail tendon from 1.7- to 35.3-mo-old (C57BL/6) male mice. Frequency histograms of D for all age groups were modeled as two normally distributed subpopulations with smaller (D(D1)) and larger (D(D2)) mean Ds, respectively. Both D(D1) and D(D2) increase from 1.6 to 4.0 mo but decrease thereafter. From tensile tests to rupture, two strain energy densities were calculated: 1) u(E) [from initial loading until the yield stress (σ(Y))], which contributes primarily to tendon resilience, and 2) u(F) [from σ(Y) through the maximum stress (σ(U)) until rupture], which relates primarily to resistance of the tendons to rupture. As measured by the normalized strain energy densities u(E)/σ(Y) and u(F)/σ(U), both the resilience and resistance to rupture increase with increasing age and peak at 23.0 and 4.0 mo, respectively, before decreasing thereafter. Multiple regression analysis reveals that increases in u(E)/σ(Y) (resilience energy) are associated with decreases in D(D1) and increases in D(D2), whereas u(F)/σ(U) (rupture energy) is associated with increases in D(D1) alone. These findings support a model where age-related variations in tendon resilience and resistance to rupture can be directed by subtle changes in the bimodal distribution of Ds.

Effects of frozen storage temperature on the elasticity of tendons from a small murine model.

The basic mechanism of reinforcement in tendons addresses the transfer of stress, generated by the deforming proteoglycan (PG)-rich matrix, to the collagen fibrils. Regulating this mechanism involves the interactions of PGs on the fibril with those in the surrounding matrix and between PGs on adjacent fibrils. This understanding is key to establishing new insights on the biomechanics of tendon in various research domains. However, the experimental designs in many studies often involved long sample preparation time. To minimise biological degradation the tendons are usually stored by freezing. Here, we have investigated the effects of commonly used frozen storage temperatures on the mechanical properties of tendons from the tail of a murine model (C57BL6 mouse). Fresh (unfrozen) and thawed samples, frozen at temperatures of -20°C and -80°C, respectively, were stretched to rupture. Freezing at -20°C revealed no effect on the maximum stress (σ), stiffness (E), the corresponding strain (ε) at σ and strain energy densities up to ε (u) and from ε until complete rupture (up). On the other hand, freezing at -80°C led to higher σ, E and u; ε and up were unaffected. The results implicate changes in the long-range order of radially packed collagen molecules in fibrils, resulting in fibril rupture at higher stresses, and changes to the composition of extrafibrillar matrix, resulting in an increase in the interaction energy between fibrils via collagen-bound PGs.

Proteomic study of calpain interacting proteins during skeletal muscle aging.

Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern. Although sarcopenia is well documented, the molecular mechanisms of this condition still remain unclear. The calcium-dependent proteolytic system is composed of calcium-dependent cysteine proteases named calpains. Calpains are involved in a large number of physiological processes such as muscle growth and differentiation, and pathological conditions such as muscular dystrophies. The aim of this study was to determine the involvement of this proteolytic system in the phenotype associated with sarcopenia by identifying key proteins (substrates or regulators) interacting with calpains during muscle aging. Immunoprecipitations coupled with proteomic analyses and protein identification by mass spectrometry have been undertaken. Reverse co-immunoprecipitation, cellular colocalisation by confocal microscopy and calpain-dependent in vitro proteolysis of several of the identified proteins have been also carried out. We identified ATP synthase subunit alpha and alpha actinin 3 as key partners of calpains during muscle aging. Such interactions would suggest that calpains are implicated in many processes altered during aging including cytoskeletal disorganisation and mitochondrial dysfunction.

Control of gene expression by steroid hormones.

The mechanism of action of steroid hormones involves their interaction with tissue-specific binding sites, and results in a precise modulation of gene expression. Both high-affinity receptors and secondary binding sites exist for steroid hormones in target tissues. Only steroid-receptor complexes were, in several cases, clearly shown to directly regulate transcription by interacting with DNA region(s) close to steroid-controlled genes. However other indications suggest that steroid hormones could also modulate transcription by altering chromatin conformation. These modifications encompass post-traductional modifications of histones and non-histone proteins, as well as changes in the pattern of histone variants. Beside transcription, there are also evidences that steroid hormones can modulate gene expression by regulating some RNA processing events. Whether high-affinity receptors or secondary binding sites directly regulate these events is not known. These observations however suggest that several levels of control might exist for steroid hormones to precisely regulate gene expression.