Modelling a ciliopathy: Ahi1 knockdown in model systems reveals an essential role in brain,retinal, and renal development

Joubert syndrome and related diseases (JSRD) are cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy, and nephronophthisis (a cystic kidney disease). Mutations in AHI1 are the most common genetic cause of JSRD, with developmental hindbrain anomalies and retinal degeneration being prominent features. We demonstrate that Ahi1, a WD40 domain-containing protein, is highly conserved throughout evolution and its expression associates with ciliated organisms. In zebrafish ahi1 morphants, the phenotypic spectrum of JSRD is modeled, with embryos showing brain, eye, and ear abnormalities, together with renal cysts and cloacal dilatation. Following ahi1 knockdown in zebrafish, we demonstrate loss of cilia at Kupffer’s vesicle and subsequently defects in cardiac left–right asymmetry. Finally, using siRNA in renal epithelial cells we demonstrate a role for Ahi1 in both ciliogenesis and cell–cell junction formation. These data support a role for Ahi1 in epithelial cell organization and ciliary formation and explain the ciliopathy phenotype of AHI1 mutations in man.     

Isoform specific phosphorylation of p53 by protein kinase CK1

The ability of three isoforms of protein kinase CK1 (α, γ₁, and δ) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1–28 sequence. Both substrates are readily phosphoylated by CK1δ and CK1α, but not by the γ isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K _m 0.82 μM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K²²¹RQK²²⁴ loop according to modeling and mutational analysis.     

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B. Received 10 August 2008; received after revision 22 October 2008; accepted 27 October 2008     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

Euler’s beta integral in Pietro Mengoli’s works

Beta integrals for several non-integer values of the exponents were calculated by Leonhard Euler in 1730, when he was trying to find the general term for the factorial function by means of an algebraic expression. Nevertheless, 70 years before, Pietro Mengoli (1626–1686) had computed such integrals for natural and half-integer exponents in his Geometriae Speciosae Elementa (1659) and Circolo(1672) and displayed the results in triangular tables. In particular, his new arithmetic–algebraic method allowed him to compute the quadrature of the circle. The aim of this article is to show how Mengoli calculated the values of these integrals as well as how he analysed the relation between these values and the exponents inside the integrals. This analysis provides new insights into Mengoli’s view of his algorithmic computation of quadratures.     

Interaction of Co, Mn, Mg and Al with d(GCCCATGGC) and d(CCGGGCCCGG): a spectroscopic study

Spectroscopic study of the interactions of metal ions, Co, Mn, Mg and Al with d(GCCCATGGGC) and d(CCGGGCCCGG) revealed the following. Metal ions Mn, Al and Mg at the lowest concentrations enhanced the t _m of oligomers, whereas Mn and Mg at higher concentrations decreased the t _m . Co enhanced the t _m of oligomers at higher concentrations. The studies have also indicated that Mn at lower concentrations displaced EtBr fluorescence, Mg and Co at moderate concentrations and Al only at higher concentrations. Addition of Co, Mn, Mg and Al altered the bands of the circulars dichroism (CD) spectra of the oligomers in a concentration-dependent manner. The CD spectra of d(GCCCATGGGC) and d(CCGGGCCCGG) indicated B and Z forms of DNA, respectively, in contrast to the A form observed in the crystal structures. Mg and Co at different ionic strength induced Z–B transition in d(CCGGGCCCGG), while Al at higher concentrations induced a Z–A transition. Mn did not induce any transition. This is the first report to show that Al causes structural transitions in sequence-specific oligomers and has strong binding ability with GC-rich euchromatin oligomers. Received 22 December 1997; received after revision 16 March 1998; accepted 16 March 1998     

<Emphasis Type="Italic">Newton's Argument for Proposition 1 of the</Emphasis> Principia

The first proposition of the Principia records two fundamental properties of an orbital motion: the Fixed Plane Property (that the orbit lies in a fixed plane) and the Area Property (that the radius sweeps out equal areas in equal times). Taking at the start the traditional view, that by an orbital motion Newton means a centripetal motion – this is a motion ``continually deflected from the tangent toward a fixed center' – we describe two serious flaws in the Principia's argument for Proposition 1, an argument based on a polygonal impulse approximation. First, the persuasiveness of the argument depends crucially on the validity of the Impulse Assumption: that every centripetal motion can be represented as a limit of polygonal impulse motions. Yet Newton tacitly takes the Impulse Assumption for granted. The resulting gap in the argument for Proposition 1 is serious, for only a nontrivial analysis, involving the careful estimation of accumulating local errors, verifies the Impulse Assumption. Second, Newton's polygonal approximation scheme has an inherent and ultimately fatal disability: it does not establish nor can it be adapted to establish the Fixed Plane Property. Taking then a different view of what Newton means by an orbital motion – namely that an orbital motion is by definition a limit of polygonal impulse motions – we show in this case that polygonal approximation can be used to establish both the fixed plane and area properties without too much trouble, but that Newton's own argument still has flaws. Moreover, a crucial question, haunted by error accumulation and planarity problems, now arises: How plentiful are these differently defined orbital motions? Returning to the traditional view, that Newton's orbital motions are by definition centripetal motions, we go on to give three proofs of the Area Property which Newton ``could have given' – two using polygonal approximation and a third using curvature – as well as a proof of the Fixed Plane Property which he ``almost could have given.' (Received August 14, 2002) Published online March 26, 2003 Communicated by G. Smith     

Functional diversity of melanopsins and their global expression in the teleost retina

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like (m) and non-mammalian-like (x) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins (opn4m-1, opn4m-2, opn4m-3, opn4x-1 and opn4x-2), each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484 nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis- and trans-isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11-cis to all-trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct “fine-tuning” of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.     

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease

The amyloid β-peptide (Aβ) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer’s disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Aβ and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Aβ can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Aβ might have a stronger (or weaker) inhibitory effect on the proteasome than does native Aβ. We therefore also investigated the proteasome inhibitory action of Aβ _1–40 (a peptide comprising the first 40 residues of Aβ) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H₂O₂ modification of Aβ _1–40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Aβ _1–40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer’s disease Received 26 September 2000; accepted 26 September 2000     

DNA synthesis in the nuclei of differentiating muscle fibers of the silkworm,Bombyx mori L.

Summary Incorporation of³H-thymidine (³HTdr) into the nuclei of myofibril-containing myofibers of larvae of the silkworm,Bombyx mori, was shown by means of light microscope (LM) and electron-microscope (EM) autoradiography. The number of DNA-synthesizing myonuclei attains 42% 12–18 h after each molt. Thus in the developing silkworm DNA replication and myofibrillogenesis are coexisting and not mutually exclusive processes as is the rule in vertebrate somatic myogenesis.     

The distribution of α2β1, α3β1 and α6β4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle

The human hair follicle is composed of different concentric compartments, which reflect different programmes of differentiation. Using monoclonal antibodies against α2β1 and α3β1 integrins we demonstrated a shift in their expression, from a basolateral distribution in the basal cells of the lower outer root sheath, to an apicolateral expression in the upper outer root sheath, as in epidermis. This shift takes place in a transition zone, localized to the midpart of the follicle. The distinct basolateral distribution of α2β1 and α3β1 integrins in the lower portion of the outer root sheath coincides with the presence of basal cell protrusions and is probably linked to the presence of the vitreous membrane which surrounds the bottom part of the anagen human hair follicle. Moreover, we showed that the expression of α6β4 integrin is discontinuous along the hair follicle and coincides with that of laminin 5. Together these results establish that within a given compartment – namely the outer root sheath – several domains can be clearly identified, which probably reflect the onset of successive differentiation pathways along the hair follicle. Received 17 January 1997; received after revision 18 February 1997; accepted 24 February 1997     

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Tautomerase superfamily members have an amino-terminal proline and a β–α–β fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF), which exhibits a phenylpyruvate tautomerase (PPT) activity. Pro-1 is a base (4-OT, CHMI, the PPT activity of MIF) or an acid (CaaD, cis-CaaD, MSAD). Components of the catalytic machinery have been identified and mechanistic hypotheses formulated. Characterization of new homologues shows that these mechanisms are incomplete. 4-OT, CaaD, cis-CaaD, and MSAD also have promiscuous activities with a hydratase activity in CaaD, cis-CaaD, and MSAD, PPT activity in CaaD and cis-CaaD, and CaaD and cis-CaaD activities in 4-OT. The shared promiscuous activities provide evidence for divergent evolution from a common ancestor, give hints about mechanistic relationships, and implicate catalytic promiscuity in the emergence of new enzymes. Received 22 May 2008; received after revision 20 June 2008; accepted 02 July 2008     

Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of ~430, ~580 and ~760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of ~430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.     

Glycoconjugates of the intestinal goblet cells of four cyprinids

The aim of this work was to show differences in the terminal and subterminal sugar composition of carbohydrate chains of glycoconjugates produced by the goblet cells of the intestines of four cyprinids. We analysed intestines of two herbivorous species – sneep and grass carp – and two omnivorous ones – chub and common carp. We compared four intestinal regions of every studied species. In every region, the presence of neutral and acidic glycoconjugates was confirmed. The smallest amount of acidic glycoconjugates was present in the second region of sneep intestine. Sulphated glycoconjugates were absent in the third and fourth region of chub intestine. Lectin histochemistry provided evidence for the presence of β-D-galactose, α-N-acetylgalactosamine, β-N-acetylglucosamine and sialic acids. Additionally, the occurrence of α-L-fucose in the goblet cells of chub, grass carp and sneep was confirmed. We tried to correlate the patern of glycoconjugate glycosylation with feeding habits of the studied fishes. Received 1 July 2002; received after revision 8 August 2002; accepted 19 August 2002 RID="*" ID="*"Corresponding author.     

Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain

Hepatitis C virus (HCV) translation initiation depends on an internal ribosome entry site (IRES). We previously identified an RNA molecule (HH363–10) able to bind and cleave the HCV IRES region. This paper characterizes its capacity to interfere with IRES function. Inhibition assays showed that it blocks IRES activity both in vitro and in a human hepatoma cell line. Although nucleotides involved in binding and cleavage reside in separate regions of the inhibitor HH363–10, further analysis demonstrated the strongest effect to be an intrinsic feature of the entire molecule; the abolishment of either of the two activities resulted in a reduction in its function. Probing assays demonstrate that HH363–10 specifically interacts with the conserved IIIf domain of the pseudoknot structure in the IRES, leading to the inhibition of the formation of translationally competent 80S particles. The combination of two inhibitory activities targeting different sequences in a chimeric molecule may be a good strategy to avoid the emergence of resistant viral variants. Received 26 July 2007; received after revision 24 September 2007; accepted 26 September 2007