Prevalence of C282Y and H63D mutations in the haemochromatosis (HFE) gene in Tunisian population

The studies of the HFE mutations: H63D and C282Y in North African populations have revealed the extreme rarity or even the absence of the C282Y mutation. We have examined 1140 chromosomes (570 Tunisian people) for the presence of the two HFE mutations by PCR-RFLP analysis. We have found that the allele frequencies are, respectively, 15.17% (+/-2.1%) for the H63D and 0.09% (+/-0.17%) for the C282Y. These results are consistent with the worldwide spread of the H63D mutation and the north European restriction of the C282Y. This study will be completed by determining whether homozygote trait for H63D and associated risk factors (beta thalassémia) can lead to iron overload in Tunisia.     

Optimisation of L-lysine production byCorynebacterium sp in fed-batch cultures

Summary The influence of initial concentration of glucose from 60 to 233 g/l on the production of L-lysine byCorynebacterium sp was studied first in batch culture. The maximum conversion rate into L-lysine was obtained at 165 g/l and the best specific production rate for L-lysine was observed at 65 g/l of glucose. In fed-batch fermentations, better conversion and the specific production rates were obtained. Maintaining of a high glucose concentration in the fed-batch technique allowed a 54% increase of the L-lysine production compared to the batch culture.     

Acceleration and Deceleration Capacity of Fetal Heart Rate in an In-Vivo Sheep Model

Background

Fetal heart rate (FHR) variability is an indirect index of fetal autonomic nervous system (ANS) integrity. FHR variability analysis in labor fails to detect early hypoxia and acidemia. Phase-rectified signal averaging (PRSA) is a new method of complex biological signals analysis that is more resistant to non-stationarities, signal loss and artifacts. It quantifies the average cardiac acceleration and deceleration (AC/DC) capacity.

Objective

The aims of the study were: (1) to investigate AC/DC in ovine fetuses exposed to acute hypoxic-acidemic insult; (2) to explore the relation between AC/DC and acid-base balance; and (3) to evaluate the influence of FHR decelerations and specific PRSA parameters on AC/DC computation.

Methods

Repetitive umbilical cord occlusions (UCOs) were applied in 9 pregnant near-term sheep to obtain three phases of MILD, MODERATE, and SEVERE hypoxic-acidemic insult. Acid-base balance was sampled and fetal ECGs continuously recorded. AC/DC were calculated: (1) for a spectrum of T values (T = 1÷50 beats; the parameter limits the range of oscillations detected by PRSA); (2) on entire series of fetal RR intervals or on “stable” series that excluded FHR decelerations caused by UCOs.

Results

AC and DC progressively increased with UCOs phases (MILD vs. MODERATE and MODERATE vs. SEVERE, p<0.05 for DC  = 2–5, and AC  = 1–3). The time evolution of AC/DC correlated to acid-base balance (0.4<<0.9, p<0.05) with the highest for . PRSA was not independent from FHR decelerations caused by UCOs.

Conclusions

This is the first in-vivo evaluation of PRSA on FHR analysis. In the presence of acute hypoxic-acidemia we found increasing values of AC/DC suggesting an activation of ANS. This correlation was strongest on time scale dominated by parasympathetic modulations. We identified the best performing parameters (), and found that AC/DC computation is not independent from FHR decelerations. These findings establish the basis for future clinical studies.     

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-α, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.The generation of recombinant polyvalent and/or polyspecific fusion proteins for use as components of novel drugs is still hindered by factors that limit their production, storage, and use, chief of which are issues related to instability and/or inadequate solubility. Here we describe a novel approach based on the use of uteroglobin (UG)³ as a skeleton for the generation of polyvalent/polyspecific recombinant proteins. Human UG is a small (15.8 kDa) globular, nonglycosylated, and homodimeric secreted protein that was discovered independently by two groups in the 1960s in rabbit uterus (1, 2), and it is the first member of a new superfamily of proteins, the so-called Secretoglobins (Scgb) (3). UG is present in the blood at a concentration of about 15 μg/ml and is found in urine and in other body fluids. The UG monomer is composed of about 70 amino acids, depending on the species, and is organized in a four α-helix secondary structure; the two subunits are joined in an anti-parallel fashion by disulfide bridges established between two highly conserved cysteine residues in amino- and carboxyl-terminal positions (4) (see Fig. 1). The exact functions of UG are not yet clear, but the protein has been reported to have anti-inflammatory properties due to its ability to inhibit the soluble phospholipase A₂. Moreover, UG contains a central hydrophobic cavity able to accommodate hydrophobic molecules such as progesterone, retinol, and prostaglandin D₂. Theoretically, this cavity could be loaded with different types of therapeutic hydrophobic substances and delivered to targets (for exhaustive reviews on UG, see Refs. 5, 6 and references therein).Open in a separate windowFIGURE 1.Central part of the figure depicts the ribbon structure of the oxidized homodimer of UG (adapted with permission from Ref. 4). A–E show the schemes of the various fusion proteins produced using UG as a central core. L19 is an scFv specific for the angiogenesis-associated FN isoform, and D2E7 is an scFv able to neutralize TNF-α.The high solubility and stability of UG to pH and temperature variations, its resistance to proteases, and its homodimeric structure prompted us to consider the protein as a candidate linker for the generation of polyvalent and polyspecific recombinant proteins. We demonstrate here that the use of UG as a linker could provide a general method for the generation of covalently linked bivalent and tetravalent antibodies, either monospecific or bispecific, as well as of different kinds of fusion proteins, which, compared with similar fusion proteins without UG, possess generally enhanced properties of solubility and stability, factors that expedite their storage and clinical use.We describe the use of UG for the production of a bivalent and tetravalent format of L19, an scFv specific for the angiogenesis-associated extra domain B (ED-B) of fibronectin (FN) (7), of an immunocytokine composed of IL2 and L19, and of a tetravalent dual specificity antibody composed of L19 and the scFv D2E7, a human antibody able to neutralize TNF-α activity (8). We report and discuss the characterization, properties, and the biological activity, both in vitro and in vivo, of these molecules.     

Studies on 1-beta-D-arabinofuranosyl-cytosine (Ara-C) resistant mutants of Chinese hamster fibroblasts. II. High resistance to Ara-C as a genetic marker for cellular hybridization

From a variety of independent Chinese hamster cell lines, stable variants resistant to 5 μg/ml of Ara-C were isolated via a single step selection; in contrast to variants selected at lower drug concentrations, the resistant clones appear to be uniformly deficient in Ara-C phosphorylation, an activity previously shown [14] to be carried out in hamster cells by a cytoplasmic dC-kinase (dC-kinase 2). These dC-kinase deficient (dCK^?) variants can be selected against because they are unable to divide in a medium containing dT (0.8 mM) and dC (0.01 mM), which supports the growth of wild type dCK⁺ cells. Plating of dCK^? cells in medium supplemented with both nucleosides yields only rare clones of pseudorevertants which escape the thymidine block through a secondary unknown defect; the growth of these clones can be prevented by further addition of dA to the selective medium. As expected from the complementation pattern for the deficient enzyme activities, hybrids between a dCK^? hamster line and TK^? lines of either mouse or hamster could be isolated in a modified HAT medium (HAT₅₀dC) containing dC and an increased dT concentration. In principle, the same selection can be used to isolate interspecific and intraspecific hybrids between Ara-C resistant variants obtained from a variety of mammalian species and azaguanine resistant lines deficient in HGPRT. The potential interest of this sytem for genetic mapping is discussed.     

Intracardiac Injection of Dental Pulp Stem Cells After Neonatal Hypoxia-Ischemia Prevents Cognitive Deficits in Rats

Neonatal hypoxia-ischemia (HI) is associated to cognitive and motor impairments and until the moment there is no proven treatment. The underlying neuroprotective mechanisms of stem cells are partially understood and include decrease in excitotoxicity, apoptosis and inflammation suppression. This study was conducted in order to test the effects of intracardiac transplantation of human dental pulp stem cells (hDPSCs) for treating HI damage. Seven-day-old Wistar rats were divided into four groups: sham-saline, sham-hDPSCs, HI-saline, and HI-hDPSCs. Motor and cognitive tasks were performed from postnatal day 30. HI-induced cognitive deficits in the novel-object recognition test and in spatial reference memory impairment which were prevented by hDPSCs. No motor impairments were observed in HI animals. Immunofluorescence analysis showed human-positive nuclei in hDPSC-treated animals closely associated with anti-GFAP staining in the lesion scar tissue, suggesting that these cells were able to migrate to the injury site and could be providing support to CNS cells. Our study evidence novel evidence that hDPSC can contribute to the recovery following hypoxia-ischemia and highlight the need of further investigation in order to better understand the exact mechanisms underlying its neuroprotective effects.     

Impact of mitochondriotropic quercetin derivatives on mitochondria

Mitochondria-targeted polyphenols are being developed with the intent to intervene on the levels of reactive oxygen species (ROS) in mitochondria. Polyphenols being more than just anti-oxidants, the interaction of these derivatives with the organelles needs to be characterised. We have studied the effects of two quercetin derivatives, 3-(4-O-triphenylphosphoniumbutyl)quercetin iodide (Q3BTPI) and its tetracetylated analogue (QTA3BTPI), on the inner membrane aspecific permeability, transmembrane voltage difference and respiration of isolated rat liver mitochondria. While the effects of low concentrations were too small to be reliably defined, when used in the 5-20 μM range these compounds acted as inducers of the mitochondrial permeability transition (MPT), an effect due to pro-oxidant activity. Furthermore, Q3BTPI behaved as an uncoupler of isolated mitochondria, causing depolarisation and stimulating oxygen consumption. When applied to tetramethylrhodamine methyl ester (TMRM)-loaded HepG2 or Jurkat cells uptake of the compounds was predictably associated with a loss of TMRM fluorescence, but there was no indication of MPT induction. A production of superoxide could be detected in some cells upon prolonged incubation of MitoSOX®-loaded cells with QTA3BTPI. The overall effects of these model mitochondriotropic polyphenols may thus differ considerably depending on whether their hydroxyls are protected or not and on the experimental system. In vivo assays will be needed for a definitive assessment of their bioactivities.     

Short acidic peptides isolated from wheat sprout chromatin and involved in the control of cell proliferation. Characterization by infrared spectroscopy and mass spectrometry

Low molecular weight peptides were isolated from the chromatin of wheat sprouts. Following gel filtration the peptide fraction shows a sharp inhibiting activity on the growth of HeLa cancer cells. Infrared (IR) spectroscopy and mass spectrometry have been utilized to characterize the wheat sprout peptides in an attempt to recognize the peptide sequence involved in the control of cell growth. The quantitative presence of a peptide with MH+=572 appears proportional to the cell growth inhibition activity. This compound has been subjected to extensive mass spectrometry analysis. The automatic computational analysis of the ions of second, third and fourth generations indicate a peptide sequence, AcHis-Asp-Ser-Glu-, that binds at the C-terminal a molecule of ethanolamine. Moreover, the results show that some sequences of the wheat sprout peptide family are present in the peptide fractions isolated from several other tissues, thus supporting the hypothesis of ubiquitous regulatory peptides.     

Structural and functional aspects of the respiratory chain of synaptic and nonsynaptic mitochondria derived from selected brain regions

Studies on brain mitochondria are complicated by the regional, cellular, and subcellular heterogeneity of the central nervous system. This study was performed using synaptic and nonsynaptic mitochondria obtained from cortex, hippocampus, and striatum of male Sprague-Dawley rats (3 months old). Ubiquinone content, detected by HPLC analysis, was about 1.5 nmol/mg protein with an approximate CoQ₉/CoQ₁₀ molecular ratio of 2:1. The activities of several respiratory chain complexes were also studied (succinate-cyt.c reductase, NADH-cyt.c reductase, succinate-DCIP, ubiquinol₂-cyt.c reductase, and cytochrome oxidase), and generally found to be higher in mitochondria from cortex than from other regions. Study of the activities of some of these enzymes vs. 1/T (Arrhenius plots) showed a straight line with an activation energy between 7 and 10 kcal/mol in all the three areas considered. Only CoQ₂H₂-cyt.c reductase activity revealed a biphasic temperature dependence. Also anisotropy (as fluorescence polarization) of the hydrophobic probe DPH showed a deviation from linearity; the break points for both enzymatic activity and anisotropy were found at about 23–24°C.     

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.