Interaction of alkylputrescines with ornithine decarboxylase from rat liver and Escherichia coli: an in vitro and in vivo study

The inhibitory effect of a series of 2-alkylputrescines on rat liver and Escherichia coli ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. At 2.5 mM concentrations, 2-methyl-, 2-propyl-, 2-butyl-, 2-pentyl- and 2-hexylputrescines were stronger inhibitors of the mammalian enzyme than putrescine. Only the higher homologues (from 2-propyl- to 2-hexylputrescine) were inhibitors of the E. coli enzyme. An analysis of the effect of increasing concentrations of the 2-alkylputrescines showed that the main difference in the behaviour of the mammalian and E. coli decarboxylases toward 2-alkylputrescines was that the former was strongly inhibited by 2-methylputrescine whereas the latter was not. 2-Alkylputrescines were found to be competitive inhibitors of both the bacterial and mammalian enzyme. The smallest Ki values (0.1 and 0.5 mM) were found for the 2-hexyl- and 2-pentylputresciens. N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescines (50 mumol per 100 g body weight) were assayed as inhibitors of thioacetamide-induced rat liver ornithine decarboxylase. N-Propylputrescine was found to be the most inhibitory (66% inhibition) and although the N-alkylputrescines were taken up by the liver, they did not inhibit the liver polyamine pools. Both putrescine and N-methylputrescine were found to stabilize the thioacetamide-induced ornithine decarboxylase at the onset of the enzyme's degradation, while 2-alkylputrescines were inhibitory under similar conditions. N-Methylputrescine induced antizyme in thioacetamide-treated rats. In thioacetamide- or dexamethasone-treated rats, 2-methylputrescine was found to be the strongest in vivo inhibitor of the liver decarboxylase. Although 2-alkylputrescines were efficiently taken up by the liver, they did not noticeably inhibit its polyamine pools. 2-methylputrescine decreased the putrescine concentration of the liver, but not its spermidine and spermine content. No induction of ornithine decarboxylase antizyme by 2-methylputrescine could be detected. The intrahepatic concentration of the latter decreased with time, very likely due to its degradation by a diamine oxidase, since the decrease was inhibited by aminoguanidine.     

No founder effect detected in Jewish Ashkenazi patients with fragile-X syndrome

Several studies on small homogenous populations suggested that fragile-X syndrome originated from a limited number of founder chromosomes. The Israeli Jewish population could serve as an adequate model for tracing a founder effect due to the unique ethnic makeup and traditional lifestyle. Furthermore, a common haplotype for Jewish Tunisian fragile X patients was recently reported. To test for a similar occurrence in the Jewish Ashkenazi population, we performed haplotype analysis of 23 fragile-X patients and 28 normal chromosomes, all Jewish Ashkenazi, using microsatellite markers within and flanking the FMR-1 gene: FRAXAC1, FRAXAC2, and DXS548. The combined triple-marker analysis identified a wide range of diverse haplotypes in patients and controls, with no distinct haplotype prevalent in the patient group. Our data suggest that no common ancestral X chromosome is associated with the fragile-X syndrome in the Israeli Jewish Ashkenazi patient population studied. These findings are in contrast to other reports on founder effect associated with fragile-X syndrome in distinct European as well as Jewish Tunisian populations. On this basis, a more complex mechanism for the development of fragile-X syndrome in the Jewish Ashkenazi population should be considered. Received: 12 May 1997 / Accepted: 24 July 1997     

Hyperpolarized Functional Magnetic Resonance of Murine Skeletal Muscle Enabled by Multiple Tracer-Paradigm Synchronizations

Measuring metabolism''s time- and space-dependent responses upon stimulation lies at the core of functional magnetic resonance imaging. While focusing on water''s sole resonance, further insight could arise from monitoring the temporal responses arising from the metabolites themselves, in what is known as functional magnetic resonance spectroscopy. Performing these measurements in real time, however, is severely challenged by the short functional timescales and low concentrations of natural metabolites. Dissolution dynamic nuclear polarization is an emerging technique that can potentially alleviate this, as it provides a massive sensitivity enhancement allowing one to probe low-concentration tracers and products in a single-scan. Still, conventional implementations of this hyperpolarization approach are not immediately amenable to the repeated acquisitions needed in real-time functional settings. This work proposes a strategy for functional magnetic resonance of hyperpolarized metabolites that bypasses this limitation, and enables the observation of real-time metabolic changes through the synchronization of stimuli-triggered, multiple-bolus injections of the metabolic tracer ¹³C₁-pyruvate. This new approach is demonstrated with paradigms tailored to reveal in vivo thresholds of murine hind-limb skeletal muscle activation, involving the conversion of ¹³C₁-pyruvate to ¹³C₁-lactate and ¹³C₁-alanine. These functional hind-limb studies revealed that graded skeletal muscle stimulation causes commensurate increases in glycolytic metabolism in a frequency- and amplitude-dependent fashion, that can be monitored on the seconds/minutes timescale using dissolution dynamic nuclear polarization. Spectroscopic imaging further allowed the in vivo visualization of uptake, transformation and distribution of the tracer and products, in fast-twitch glycolytic and in slow-twitch oxidative muscle fiber groups. While these studies open vistas in time and sensitivity for metabolic functional magnetic resonance studies in muscle, the simplicity of our approach makes this technique amenable to a wide range of functional metabolic tracer studies.     

Analyse chromosomique des œufs humains non clivés après injection intracytoplasmique de spermatozoïde

The results of intracytoplasmic sperm injection still need to be assessed concerning both its efficiency and its possible risks for the children to be born. Cytogenetical analysis of uncleaved oocytes after ICSI can give different types of information. It can help in determining the cause of the failure, checking the injection and specifying the development stage of the spermatozoa and its possible abnormalities. It also allows an evaluation of the possible chromosome abnormalities induced in the oocyte and subsequently of the safety of the procedure for the oocyte itself. After conventional in vitro fertilization (IVF) the main cause of the lack of cleavage is the total absence of fertilization but the premature condensation of the spermatozoa chromosomes (PCC) is observed in about 10% of the cases. This might be different after ICSI because of the procedure itself or because of the sperm defect which requires ICSI to achieve fertilization. We studied ICSI failures during two periods: the first one started at the beginning of the use of the technique in our laboratory and the second one followed, using a different technique (pushing the spermatozoa further in the oocyte by aspirating more vigourously oocyte cytoplasm). The fertilization rates were 15% and 54% in the two periods. In the first period the main cause of the failure was the total absence of evolution of the spermatozoa in the oocyte and it represented only 33% of the cases of the second period. In the second period the incidence of PCC increased and the total absence of evolution was less frequent while the incidence of chromosome fragmentation in the oocyte remained high. Our results suggest that the technique used for ICSI is very important to avoid the secondary extrusion of the spermatozoa. A possible increase of oocyte chromosome breakage has to be confirmed.     

The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice

ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1^−/− mice have reduced viability and fail to thrive early after birth. Male Ube2j1^−/− mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1^−/− elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.     

Crystal Structures of a Group II Chaperonin Reveal the Open and Closed States Associated with the Protein Folding Cycle

Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.     

Nonparametric estimation in an “illness‐death” model when all transition times are interval censored

We develop nonparametric maximum likelihood estimation for the parameters of an irreversible Markov chain on states from the observations with interval censored times of 0 → 1, 0 → 2 and 1 → 2 transitions. The distinguishing aspect of the data is that, in addition to all transition times being interval censored, the times of two events (0 → 1 and 1 → 2 transitions) can be censored into the same interval. This development was motivated by a common data structure in oral health research, here specifically illustrated by the data from a prospective cohort study on the longevity of dental veneers. Using the self‐consistency algorithm we obtain the maximum likelihood estimators of the cumulative incidences of the times to events 1 and 2 and of the intensity of the 1 → 2 transition. This work generalizes previous results on the estimation in an “illness‐death” model from interval censored observations.     

Mutations in FYCO1 cause autosomal-recessive congenital cataracts

Congenital cataracts (CCs), responsible for about one-third of blindness in infants, are a major cause of vision loss in children worldwide. Autosomal-recessive congenital cataracts (arCC) form a clinically diverse and genetically heterogeneous group of disorders of the crystalline lens. To identify the genetic cause of arCC in consanguineous Pakistani families, we performed genome-wide linkage analysis and fine mapping and identified linkage to 3p21-p22 with a summed LOD score of 33.42. Mutations in the gene encoding FYVE and coiled-coil domain containing 1 (FYCO1), a PI(3)P-binding protein family member that is associated with the exterior of autophagosomes and mediates microtubule plus-end-directed vesicle transport, were identified in 12 Pakistani families and one Arab Israeli family in which arCC had previously been mapped to the overlapping CATC2 region. Nine different mutations were identified, including c.3755 delC (p.Ala1252AspfsX71), c.3858_3862dupGGAAT (p.Leu1288TrpfsX37), c.1045 C>T (p.Gln349X), c.2206C>T (p.Gln736X), c.2761C>T (p.Arg921X), c.2830C>T (p.Arg944X), c.3150+1 G>T, c.4127T>C (p.Leu1376Pro), and c.1546C>T (p.Gln516X). Fyco1 is expressed in the mouse embryonic and adult lens and peaks at P12d. Expressed mutant proteins p.Leu1288TrpfsX37 and p.Gln736X are truncated on immunoblots. Wild-type and p.L1376P FYCO1, the only missense mutant identified, migrate at the expected molecular mass. Both wild-type and p. Leu1376Pro FYCO1 proteins expressed in human lens epithelial cells partially colocalize to microtubules and are found adjacent to Golgi, but they primarily colocalize to autophagosomes. Thus, FYCO1 is involved in lens development and transparency in humans, and mutations in this gene are one of the most common causes of arCC in the Pakistani population.