Thyroid remnant dose: 124I-PET/CT dosimetric comparison of rhTSH versus thyroid hormone withholding before radioiodine remnant ablation in differentiated thyroid cancer.

AIM: Recombinant human thyroid-stimulating hormone (rhTSH) recently was approved as an alternative to thyroid hormone withholding (THW) to elevate TSH for thyroid remnant ablation in differentiated thyroid carcinoma patients. High ablation success rates are reported with diverse rhTSH-aided (131)I activities. Improved renal function causes approximately 50% faster radioiodine clearance under euthyroidism versus hypothyroidism. Knowledge of comparative remnant radioiodine kinetics, particularly the remnant radiation dose in Gy/GBq of administered (131)I activity (RDpA), could assist in choosing rhTSH-aided ablative activities. MATERIAL AND METHODS: To compare the RDpA, determined through (124)I-positron emission tomography/computed tomography (PET/CT), under the two stimulation methods, we retrospectively divided into two groups 55 consecutive totally-thyroidectomized, radioiodine-naïve patients. The rhTSH group (n=16) received (124)I on thyroid hormone, 24 h after two consecutive daily intramuscular injections of rhTSH, 0.9 mg. The THW group (n=39) received (124)I after weeks-long THW, when serum TSH first measured > or = 25 mIU/L. We performed PET investigations 4 h, 24 h, 48 h, 72 h and 96 h and PET/CT 25 h after (124)I administration. RESULTS: Median stimulated serum thyroglobulin was 15 times higher (p=0.023) and M1 disease almost twice as prevalent (p=0.05) in rhTSH versus THW patients. Mean+/-standard deviation RDpA was statistically equivalent between the groups: rhTSH, 461+/-600 Gy/GBq, THW, 302+/-329 Gy/GBq, two-sided p=0.258. CONCLUSIONS: rhTSH or THW deliver statistically equivalent radiation doses to thyroid remnant and may be chosen based on safety, quality-of-life, convenience and pharmacoeconomic factors. Institutional fixed radioiodine activities formulated for use with THW need not be adjusted for rhTSH-aided ablation.

CRB1 has a cytoplasmic domain that is functionally conserved between human and Drosophila.

Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies, ranging from retinitis pigmentosa to Leber congenital amaurosis. The CRB1 gene is expressed specifically in human retina and brain and encodes a protein homologous to the Drosophila Crumbs protein. In crumbs mutant embryos apico-basal polarity of epithelial cells is lost, leading to widespread epidermal cell death. The small cytoplasmic domain of Crumbs organizes an intracellular protein scaffold that defines the assembly of a continuous zonula adherens. The crumbs mutant phenotype can be partially rescued by expression of just the membrane-bound cytoplasmic domain, and overexpression of this domain in a wild-type background results in a multilayered epidermis. A striking difference between CRB1 and Crumbs was that the latter contains a transmembrane region and a 37 amino acid cytoplasmic domain. Here we describe an alternative splice variant of human CRB1 that encodes a cytoplasmic domain 72% similar to that of Drosophila Crumbs. Two intracellular subdomains that are necessary for function in Drosophila are absolutely conserved. Rescuing and overexpression studies in Drosophila show that the cytoplasmic domains are functionally related between these distant species. This suggests that CRB1 organizes an intracellular protein scaffold in the human retina. Human homologues of proteins binding to Crumbs may be part of this complex and represent candidate genes for retinal dystrophies.

Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.

The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.

G protein signaling and asymmetric cell division.

Asymmetric cell division depends on the polarization of the dividing cell for the correct alignment of the mitotic spindle and the localization of cytoplasmic determinants. Receptor-independent activation of heterotrimeric G proteins by the Drosophila GoLoco protein Partner of Inscuteable seems to represent a novel mechanism to control these events.

Zonula adherens formation in Caenorhabditis elegans requires dlg-1, the homologue of the Drosophila gene discs large.

The correct assembly of junction components, such as E-cadherin and beta-catenin, into the zonula adherens is fundamental for the function of epithelia, both in flies and in vertebrates. In C. elegans, however, the cadherin-catenin system is not essential for general adhesion, raising the question as to the genetic basis controlling junction morphogenesis in nematodes. Here we show that dlg-1, the C. elegans homologue of the Drosophila tumour-suppressor gene discs-large, plays a crucial role in epithelial development. DLG-1 is restricted to adherens junctions of all embryonic epithelia, which contrasts with the localisation of the Drosophila and vertebrate homologues in septate and tight junctions, respectively. Proper localisation of DLG-1 requires the basolateral LET-413 protein, but is independent of the cadherin-catenin system. Embryos in which dlg-1 activity was eliminated by RNA-mediated interference fail to form a continuous belt of junction-associated antigens and arrest development. Loss of dlg-1 activity differentially affects localisation of proteins normally enriched apically to the zonula adherens. While the distribution of an atypical protein kinase C (PKC-3) and other cytoplasmic proteins (PAR-3, PAR-6) is not affected in dlg-1 (RNAi) embryos, the transmembrane protein encoded by crb-1, the C. elegans homologue of Drosophila crumbs, is no longer concentrated in this domain. In contrast to Drosophila, however, crb-1 and a second crb-like gene are not essential for epithelial development in C. elegans. Together the data indicate that several aspects of the spatial organisation of epithelial cells and its genetic control differ between flies, worms, and vertebrates, while others are conserved. The molecular nature of DLG-1 makes it a likely candidate to participate in the organisation of a protein scaffold that controls the assembly of junction components into the zonula adherens.

Epithelial morphogenesis: filopodia at work.

Spreading and fusion of epithelial sheets are conserved morphogenetic mechanisms that help shape embryos and tissues. Recent findings suggest that the formation of dynamic filopodia at the leading front of the epithelia plays a critical role in regulating cell movement and recognition during these processes.

Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts.

The establishment and maintenance of polarity is of fundamental importance for the function of epithelial and neuronal cells. In Drosophila, the multi-PDZ domain protein Bazooka (Baz) is required for establishment of apico-basal polarity in epithelia and in neuroblasts, the stem cells of the central nervous system. In the latter, Baz anchors Inscuteable in the apical cytocortex, which is essential for asymmetric localization of cell fate determinants and for proper orientation of the mitotic spindle. Here we show that Baz directly binds to the Drosophila atypical isoform of protein kinase C and that both proteins are mutually dependent on each other for correct apical localization. Loss-of-function mutants of the Drosophila atypical isoform of PKC show loss of apico-basal polarity, multilayering of epithelia, mislocalization of Inscuteable and abnormal spindle orientation in neuroblasts. Together, these data provide strong evidence for the existence of an evolutionary conserved mechanism that controls apico-basal polarity in epithelia and neuronal stem cells. This study is the first functional analysis of an atypical protein kinase C isoform using a loss-of-function allele in a genetically tractable organism.

Control of epithelial cell shape and polarity.

The polarised character of a cell is often obvious from its shape and is largely dependent on the actin cytoskeleton and the membrane-associated cell cortex---a dense network comprising spectrin and other related proteins. Spatially and functionally distinct protein scaffolds, assembled from transmembrane and cytoplasmic proteins, provide the cues for polarisation. Recent data have provided new insights into the molecular nature of these cues and the mechanisms by which they may be translated into a polarised phenotype.

Preparation of 124I solutions after thermodistillation of irradiated 124TeO2 targets.

The 4.15-d radionuclide 124I is produced via the nuclear reaction 124Te(d, 2n) 124I by irradiation of 96% enriched 124TeO2 with 14 MeV deuterons, followed by thermodistillation. In order to minimise the loss of 124I, the quartz distillation tube was fitted to a stainless steel helix capillary trap directly behind the end of the furnace. Using this device, distillation yields of more than 80% were routinely obtained, and the activity was concentrated in markedly less than 100 microL solution. The 124I produced by this method proved to be useful for labelling proteins and IUdR.

Imaging brain tumor proliferative activity with [124I]iododeoxyuridine.

Iododeoxyuridine (IUdR) uptake and retention was imaged by positron emission tomography (PET) at 0-48 min and 24 h after administration of 28.0-64.4 MBq (0.76-1.74 mCi) of [124I]IUdR in 20 patients with brain tumors, including meningiomas and gliomas. The PET images were directly compared with gadolinium contrast-enhanced or T2-weighted magnetic resonance images. Estimates for IUdR-DNA incorporation in tumor tissue (Ki) required pharmacokinetic modeling and fitting of the 0-48 min dynamically acquired data to correct the 24-h image data for residual, nonincorporated radioactivity that did not clear from the tissue during the 24-h period after IUdR injection. Standard uptake values (SUVs) and tumor:brain activity ratios (Tm:Br) were also calculated from the 24-h image data. The Ki, SUV, and Tm/Br values were related to tumor type and grade, tumor labeling index, and survival after the PET scan. The plasma half-life of [124I]IUdR was short (2-3 min), and the arterial plasma input function was similar between patients (48 +/- 12 SUV*min). Plasma clearance of the major radiolabeled metabolite ([124I]iodide) varied somewhat between patients and was markedly prolonged in one patient with renal insufficiency. It was apparent from our analysis that a sizable fraction (15-93%) of residual nonincorporated radioactivity (largely [124I]iodide) remained in the tumors after the 24-h washout period, and this fraction varied between the different tumor groups. Because the SUV and Tm:Br ratio values reflect both IUdR-DNA incorporated and exchangeable nonincorporated radioactivity, any residual nonincorporated radioactivity will amplify their values and distort their significance and interpretation. This was particularly apparent in the meningioma and glioblastoma multiforme groups of tumors. Mean tumor Ki values ranged between 0.5 +/- 0.9 (meningiomas) and 3.9 +/- 2.3 microl/min/g (peak value for glioblastoma multiforme, GBM). Comparable SUV and Tm:Br values at 24 h ranged from 0.13 +/- 0.03 to 0.29 +/- 0.19 and from 2.0 +/- 0.6 to 6.1 +/- 1.5 for meningiomas and peak GBMs, respectively. Thus, the range of values was much greater for Ki (approximately 8-fold) compared with that for SUV (approximately 2.2-fold) and Tm:Br (approximately 3-fold). The expected relationships between Ki, SUV, and Tm:Br and other measures of tumor proliferation (tumor type and grade, labeling index, and patient survival) were observed. However, greater image specificity and significance of the SUV and Tm:Br values would be obtained by achieving greater washout and clearance of the exchangeable fraction of residual (background) radioactivity in the tumors, i.e., by increased hydration and urinary clearance and possibly by imaging later than 24 h after [124I]IUdR administration.

A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila.

BACKGROUND: Specialised cell junctions in epithelia serve as cell-cell adhesion sites and thus contribute to the maintenance of tissue integrity. The Drosophila gene crumbs encodes a transmembrane protein that is required for the biogenesis of the zonula adherens, a belt-like structure encircling the apex of epithelial cells. As previously shown, expression of just the short membrane-bound cytoplasmic domain is sufficient to rescue major defects associated with the loss of crumbs function. RESULTS: The cytoplasmic domain of Crumbs is highly conserved in two putative crumbs homologues in Caenorhabditis elegans. To assess the significance of conserved residues, various point mutations and deletions were introduced into this region. Two functional domains were revealed, an amino-terminal region and the carboxy-terminal amino acids EERLI. Both are necessary for rescue of the crumbs phenotype. The EERLI motif interacts with Discs Lost, a cytoplasmic protein containing PDZ domains. Overexpression of the Crumbs cytoplasmic domain induces a transition from the single-layered epithelium to a multilayered tissue. This transition is associated with redistribution of the Drosophila homologue of the cell adhesion molecule E-cadherin, and depends on the presence of the EERLI motif. CONCLUSIONS: We propose a model in which the interaction of the Crumbs carboxyl terminus with Discs Lost organises a membrane-associated protein complex in the apical cytocortex of epithelial cells. This scaffold mediates the localisation and stabilisation of the zonula adherens component DE-cadherin, a crucial component for the maintenance of epithelial cell polarity and tissue integrity.

Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts.

Asymmetric cell division generates daughter cells with different developmental fates from progenitor cells that contain localized determinants. During this division, the asymmetric localization of cell-fate determinants and the orientation of the mitotic spindle must be precisely coordinated. In Drosophila neuroblasts, inscuteable controls both spindle orientation and the asymmetric localization of the cell-fate determinants Prospero and Numb. Inscuteable itself is localized in an apical cortical crescent and thus reflects the intrinsic asymmetry of the neuroblast. Here we show that localization of Inscuteable depends on Bazooka, a protein containing three PDZ domains with overall sequence similarity to Par-3 of Caenorhabditis elegans. Bazooka and Inscuteable form a complex that also contains Staufen, a protein responsible for the asymmetric localization of prospero messenger RNA. We propose that, after delamination of the neuroblast from the neuroepithelium, Bazooka provides an asymmetric cue in the apical cytocortex that is required to anchor Inscuteable. As Bazooka is also responsible for the maintenance of apical-basal polarity in epithelial tissues, it may be the missing link between epithelial polarity and neuroblast polarity.

bloated tubules (blot) encodes a Drosophila member of the neurotransmitter transporter family required for organisation of the apical cytocortex.

We have identified a novel member of the vertebrate sodium- and chloride-dependent neurotransmitter symporter family from Drosophila melanogaster. This gene, named bloated tubules (blot), shows significant sequence similarity to a subgroup of vertebrate orphan transporters. blot transcripts are maternally supplied and during embryogenesis exhibit a complex and dynamic pattern in a subset of ectodermally derived epithelia, notably in the Malpighian tubules, and in the nervous system. Animals mutant for this gene are larval lethals, in which the Malpighian tubule cells are distended with an enlarged and disorganised apical surface. Embryos lacking the maternal component of blot expression die during early stages of development. They show an inability to form actin filaments in the apical cortex, resulting in impaired syncytial nuclear divisions, severe defects in the organisation of the cortical cytoskeleton, and a failure to cellularise. For the first time, a neurotransmitter transporter-like protein has been implicated in a function outside the nervous system. The isolation of blot thus provides the basis for an analysis of the relationship between the function of this putative transporter and epithelial morphogenesis.

Drosophila morphogenesis: orchestrating cell rearrangements.

Changes in shape of individual cells need to be coordinated to generate the movements of cell groups and sheets that are so important in morphogenesis. Recent results have shown that, during Drosophila gastrulation, multiple signalling pathways act to orchestrate the complex cell rearrangements.

Positive and negative control of Serrate expression during early development of the Drosophila wing.

The product of the Drosophila gene Serrate acts as a short-range signal during wing development to induce the organising centre at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. Regulatory elements reflecting the early Serrate expression in the dorsal compartment of the wing disc have recently been confined to a genomic fragment in the 5'-upstream region of the gene. Here we present data to suggest that this fragment responds to various positive and negative inputs required for the early Serrate expression. First, activation and maintenance of expression in the dorsal compartment of the wing discs of second and early third instar larvae depends on apterous, as revealed by reporter gene expression in discs either lacking or ectopically expressing apterous. Second, transcriptional downregulation during third larval instar is mediated by hiiragi. Finally, this regulatory element responds to Delta signalling in a nonautonomous way to maintain Serrate expression along the dorsal margin. The results clearly show that some of the previously described transactivators of Serrate protein expression, e.g. fringe, act on elements required for later aspects of Serrate expression.

Dissection of cis-regulatory elements of the Drosophila gene Serrate.

The Drosophila gene Serrate encodes a membrane spanning protein, which is expressed in a complex pattern during embryogenesis and larval stages. Loss of Serrate function leads to larval lethality, which is associated with several morphogenetic defects, including the failure to develop wings and halteres. Serrate has been suggested to act as a short-range signal during wing development. It is required for the induction of the organising centre at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. In order to understand the regulatory network required to control the spatially and temporally dynamic expression of Serrate, we analysed its cis-regulatory elements by fusing various genomic fragments upstream of the reporter gene lacZ. Enhancer elements reflecting the expression pattern of endogenous Serrate in embryonic and postembryonic tissues could be confined to 26 kb of genomic DNA, including 9 kb of transcribed region. Expression in some embryonic tissues is under the control of multiple enhancers located in the 5' region and in intron sequences. The data presented here provide the tools to unravel the genetic network which regulates Serrate during different developmental stages in diverse tissues.

Radiosynthesis and quality assurance of 5-[124I]Iodo-2'-deoxyuridine for functional PET imaging of cell proliferation.

5-[124I]Iodo-2'-deoxyuridine ([124I]IUdR) was routinely produced by direct electrophilic labelling of 2'-deoxyuridine with 124I of high specific activity (12 Ci/micromol) in an Iodogen-coated ReactiVial, followed by purification on a Sep-Pak C-18 cartridge. The radiochemical purity was determined by TLC on a Silicagel-60 plate and by reverse-phase HPLC on a RP-18 column. Based upon 45 syntheses, the yield ranged from 45% to 65%. The radiochemical impurity of [124I]IUdR was determined at 2.9% by TLC (mainly iodate) and 4.3% by HPLC. The chemical stability of the solvated formulation allowed a time window of 2 days following end of synthesis (EOS) for chemical application, based upon the required 95% radiochemical purity grade of [124I]IUdR. The labelled compound was routinely used for the clinical determination of cell proliferation in glioma patients by positron emission tomography.

Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka.

BACKGROUND: The orientation of the mitotic spindle influences the asymmetric distribution of cytoplasmic determinants and the positioning of the sibling cell, and therefore has important influences on cell-fate determination and patterning of the embryo. Both the establishment of an axis of polarity and the adjustment of this axis with respect to the coordinates of the embryo have to be controlled. None of the genes identified so far that are involved in these processes seems to have been conserved between flies and nematodes. RESULTS: Here, we show that the bazooka gene encodes a protein with three putative protein-interaction motifs known as PDZ domains and is the first Drosophila representative of the par gene family of Caenorhabditis elegans, members of which are required for establishment of anterior-posterior polarity of the nematode embryo. The bazooka RNA and protein were found to be restricted to the apical cortical cytoplasm of epithelial cells and neuroblasts. Embryos that were mutant for bazooka frequently failed to coordinate the axis of cell polarity with that of the embryo. This was manifested as defective spindle orientation and mispositioning of the daughter cell after division. CONCLUSIONS: The Drosophila gene bazooka is likely to be part of a regulatory mechanism required to coordinate the axis of polarity of a cell with that of the embryo. The PDZ domains of Bazooka provide several protein-protein interfaces, which possibly participate in the assembly of a multiprotein complex at the apical pole.

Drosophila morphogenesis: movements behind the edge.

Coordinated cell movements during development require extensive exchange of information between the cells involved. Recent results suggest a connection between two signalling pathways during dorsal closure in the Drosophila embryo.