Gene dosage effect for human triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in partial trisomy 12p13 and trisomy 18p

Summary An 8-year-old girl with profound mental retardation and a neurologic syndrome associated with morphologic abnormalities was found to have a supernumerary small submetacentric chromosome. Several members of her family carried a balanced translocation t(12;18)(p12;q11), and the child's karyotype could be explained by 3:1 maternal segregation (tertiary trisomy). The proband was trisomic for 12p13 and 18p. A gene dosage effect was demonstrated for triosephosphate isomerase and glyceraldehyde-3-phosphate in erythrocytes and leukocytes allowing us to assign the corresponding loci to the tip of the chromosome 12 short arm.     

Binding of carotenoids on reaction centers from Rhodopseudomonas sphaeroides R 26

The carotenoid-less reaction centers isolated from Rhodopseudomonas sphaeroides (strain R 26) bind pure all-trans spheroidene as well as spheroidenone in a nearly 1:1 molar ratio with respect to P-870. Neither β-carotene nor spirilloxanthin, both absent from wild-type Rps. sphaeroides, could be bound in appreciable amounts. Resonance Raman spectra of the carotenoidreaction center complex indicate that the carotenoid is bound as a cis isomer, its conformation being very close, although probably not identical, to that assumed by the carotenoid in the wild-type reaction centers. The electronic absorption spectra of the carotenoid-reaction center complexes are in good agreement with such a interpretation. When bound to the R 26 reaction centers, spheroidene displays light-induced absorbance changes identical in peak wavelengths and comparable in amplitudes to those observed in the wild-type reaction centers. Thus the binding of the carotenoid to the R 26 reaction centers most likely occurs at the same proteic site as in the wild-type reaction centers. This site shows selectivity towards the nature of carotenoids, and has the same sterical requirement as in the wild type, leading to the observed all-trans to cis isomerisation.     

THE REPRODUCTIVE RELATIONSHIPS OF DROSOPHILA SECHELLIA WITH D. MAURITIANA,D. SIMULANS,AND D. MELANOGASTER FROM THE AFROTROPICAL REGION

Hybridization tests among the four sibling species of the Drosophila melanogaster complex were made to determine the reproductive status of the recently discovered D. sechellia (which is endemic to a few islands and islets of the Seychelles archipelago) with regard to its three close relatives, D. mauritiana (endemic to Mauritius) and Afrotropical strains of the two cosmopolitan species D. melanogaster and D. simulans. Interstrain variation in the ability to hybridize with other species was also analyzed for D. melanogaster and D. simulans. D. mauritiana and D. simulans appear to be more weakly isolated from each other than either species is from D. sechellia. A striking unilateral mating success is observed in the cross of D. sechellia with D. simulans. The most extreme isolation is between D. melanogaster and its three siblings. Variation in the ability of strains to hybridize is observed in heterospecific crosses between D. simulans and either D. melanogaster or D. mauritiana.     

Interpretation of the Repetitive Firing of Nerve Cells

Eccentric cells of Limulus respond with repetitive firing to sustained depolarizing currents. Following stimulation with a step of current, latency is shorter than first interval and later intervals increase progressively. A shock of intensity twice threshold can evoke firing 25 msec. after an impulse. But in the same cell, a current step twice rheobase evokes a second impulse more than 50 msec. after the first, and current intensity must be raised to over five times rheobase to obtain a first interval of about 25 msec. Repetitive firing was evoked by means of trains of shocks. With stimuli of moderate intensity, firing was evoked by only some of the shocks and intervals between successive impulses increased with time. This is ascribed to accumulation of refractoriness with successive impulses. Higher frequencies of firing are obtained with shocks of intensity n x threshold than with constant currents of intensity n x rheobase. It is concluded that prolonged currents depress the processes leading to excitation and that (in the cells studied) repetitive firing is controlled both by the after-effects of firing (refractoriness) and by the depressant effects of sustained stimuli (accommodation). Development of subthreshold "graded activity" is an important process leading to excitation of eccentric cells, but is not the principal factor determining frequency of firing in response to constant currents.     

ElrA binding to the 3'UTR of cyclin E1 mRNA requires polyadenylation elements

The early cell divisions of Xenopus laevis and other metazoan embryos occur in the presence of constitutively high levels of the cell cycle regulator cyclin E1. Upon completion of the 12th cell division, a time at which many maternal proteins are downregulated by deadenylation and destabilization of their encoding mRNAs, maternal cyclin E1 protein is downregulated while its mRNA is polyadenylated and stable. We report here that stable polyadenylation of cyclin E1 mRNA requires three cis-acting elements in the 3′ untranslated region; the nuclear polyadenylation sequence, a contiguous cytoplasmic polyadenylation element and an upstream AU-rich element. ElrA, the Xenopus homolog of HuR and a member of the ELAV gene family binds the cyclin E1 3′UTR with high affinity. Deletion of these elements dramatically reduces the affinity of ElrA for the cyclin E1 3′UTR, abolishes polyadenylation and destabilizes the mRNA. Together, these findings provide compelling evidence that ElrA functions in polyadenylation and stabilization of cyclin E1 mRNA via binding these elements.     

Missense mutations of dual oxidase 2 (DUOX2) implicated in congenital hypothyroidism have impaired trafficking in cells reconstituted with DUOX2 maturation factor

Dual oxidase 2 (DUOX2), a reduced NAD phosphate:O2 oxidoreductase flavoprotein, is a component of the thyrocyte H2O2 generator required for hormone synthesis at the apical plasma membrane. We recently identified a specific DUOX2 maturation factor (DUOXA2) that is necessary and sufficient for expression of functional DUOX2 in mammalian cell lines. We have now used a DUOXA2 reconstituted system to provide the first characterization of natural DUOX2 missense variants (Q36H, R376W, D506N) at the molecular level, analyzing their impact on H2O2 generation, trafficking, stability, folding, and DUOXA2 interaction. The Q36H and R376W mutations completely prevent routing of DUOX2 to the cell surface. The mutant proteins are predominantly present as core N-glycosylated, thiol-reduced folding intermediates, which are retained by the quality control system within the endoplasmic reticulum (ER) as indicated by increased complexation with the lectin calnexin. D506N displays a partial deficiency phenotype with reduced surface expression of a mutant protein with normal intrinsic activity in generating H2O2. D506N N-glycan moieties are not subject to normal modification in the Golgi apparatus, suggesting that nonnative protein can escape the quality control in the ER. Oxidative folding of DUOX2 in the ER appears to be the rate-limiting step in the maturation of DUOX2, but is not facilitated by DUOXA2. Rather, DUOXA2 allows rapid ER exit of folded DUOX2 or enhanced degradation of mutant DUOX2 proteins not competent for ER exit. DUOXA2 may thus be part of a secondary quality control system specific for DUOX2.     

Calcium is transported into the lumen of pig thyroid follicles by fluid phase basolateral to apical transcytosis

The lumen of thyroid follicles contains a high concentration of thyroglobulin, the thyroid prohormone and a high concentration of calcium (Ca²⁺). As thyroglobulin binds Ca²⁺, intraluminal Ca²⁺ is expected to be in free and protein-bound forms. In the present work, we have investigated the mechanism(s) by which Ca²⁺ could enter the lumen of thyroid follicles. ⁴⁵Ca²⁺ uptake studies were carried out on reconstituted pig thyroid follicles (RTF) and pig thyroid cell monolayers (TCM) in primary culture, representing experimental systems with two compartments (cells + lumina) and one compartment, respectively. ⁴⁵Ca²⁺ accumulation in RTF was rapid during the first hour of incubation and then slowly increased. Analysis of the uptake data with a “two compartments” model gave two kinetic constant values: k_-1 = 1.71 ± 0.28 hr^-1 and k_-2 = 0.20 ± 0.05 hr^-1 (n = 10). The slow uptake process accounted for 20–50% of the total RTF-associated Ca²⁺ after 24 hr. ⁴⁵Ca²⁺ uptake by TCM was rapid and reached a stable level within 1–2 hr. Experimental data fitted with a “single compartment” model and gave a k_-1 value of 1.64 ± 0.15 hr^-1 (n = 10) which was not statistically different from the k_-1 obtained for ⁴⁵Ca²⁺ uptake by RTF. We then compared the kinetics of ⁴⁵Ca²⁺ uptake by RTF with the kinetics of transport of fluid phase markers: [¹⁴C]-sucrose and Lucifer Yellow from the medium to the lumen of RTF. [¹⁴C]-sucrose and Lucifer Yellow uptakes by RTF appeared as slow processes compatible with the entry in a single compartment with k_-1 values of 0.32 ± 0.06 hr^-1 (n = 3) and 0.23 ± 0.015 hr^-1 (n = 3), respectively. These values were not significantly different from the k_-2 value obtained for ⁴⁵Ca²⁺ uptake by RTF. These data suggest that thyroid follicles would possess two independent Ca²⁺ compartments: cells and lumen, and that the entry of Ca²⁺ into the lumen of follicles probably could take place by fluid phase basolateral to apical transcytosis. J. Cell. Physiol. 171:43–51, 1997. © 1997 Wiley-Liss, Inc.     

Evidence for participation of the epidermis in the deposition of superficial layer of scales in zebrafish (Danio rerio): A SEM and TEM study

Comparative studies on scale structure and development in bony fish have led to the hypothesis that elasmoid scales in teleosts could be dental in origin. The present work was undertaken to determine whether the scales in zebrafish (Danio rerio), a species widely used in genetics and developmental biology, would be an appropriate focus for further studies devoted to the immunodetection of dental components or to the detection of the expression of genes coding for various dental proteins in fish scales. The superficial region of mature and experimentally regenerated scales and its relationships to the epidermal cover were studied in adult zebrafish using scanning (SEM) and transmission (TEM) electron microscopy. The elasmoid scales are relatively large, thin, and are located in the upper region of the dermis, close to the epidermis. In adults, the surface of the posterior region appears smooth at the SEM level and is entirely covered by the epidermis. During regeneration, the relationship of the epidermal cover to the scale surface is established within 4 days. This interface is easier to study in regenerating than in mature scales because the former are poorly mineralized. TEM revealed that: (1) the epidermis is in direct contact with the scale surface, from which it is separated only by a basement membrane-like structure, (2) there are no dermal elements at the scale surface except at the level of grooves issuing from the focus and crossing the scale surface radially, (3) the mineral crystals located in this superficial region are perpendicular to the scale surface, whereas those located deeper within the collagenous scale matrix are randomly disposed, and (4) when decalcified, the matrix of the superficial region of the scale appears devoid of collagen fibrils but contains thin electron-dense granules, some of which are arranged into layers. The continuous epidermal covering, the absence of dermal elements, as well as the fine structure of the matrix and its type of mineralization, strongly suggest that epidermal products, possibly enamel-like proteins, are deposited at the scale surface and contribute to the thickening of the upper layer in zebrafish scales. J. Morphol. 231:161–174, 1997. © 1997 Wiley-Liss, Inc.     

Structural and functional analysis of SGT1-HSP90 core complex required for innate immunity in plants

SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization.