Review of referrals for the FISH detection of Williams syndrome highlights the importance of testing in supravalvular aortic stenosis/pulmonary stenosis.
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Adolescent↗
Biomedical subjects
L Robson
Publications and source records attributed to L Robson.
Routine fluorescence in situ hybridization with the MLL probe does not reliably detect two separate signals on one chromosome 11 in patients with trisomy 11.
Trisomy 11 is considered to be a rare cytogenetic abnormality in myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). Duplication of the MLL gene (localized to 11q23) has been found on one chromosome 11 in patients with trisomy 11, detected by DNA techniques. We investigated copy number of MLL in seven patients with trisomy 11 to see if duplication could be assessed by the detection of two separate signals on fluorescence in situ hybridization (FISH). If so, FISH could provide a quick easy screen of MLL status in routine referrals. The diagnostic bone marrow aspirate showed trisomy 11 in five adult patients with MDS/AML as part of a complex karyotype and in two children with acute lymphoblastic leukemia (ALL) as part of a hyperdiploid karyotype. Fluorescence in situ hybridization utilized the suspensions remaining after the cytogenetic harvest. Two FISH probes were used on the adult patients (MLL - Oncor and Vysis), and one (Vysis) for the two children with ALL. Analysis showed that the proximity of the two putative hybridization signals made it very difficult to unambiguously see two separate signals. The hybridisations (Oncor probe) were convincing of MLL duplication (namely two distinct signals) in only one patient, but this was not borne out with the other MLL probe (Vysis). We conclude that conventional FISH with MLL probe is not suited to act as a screen for MLL duplication in patients with trisomy 11.
Adult↗
Na+-alanine uptake activates a Cl- conductance in frog renal proximal tubule cells via nonconventional PKC.
Hyposmotically induced swelling of frog renal proximal tubule cells activates a DIDS-sensitive, outwardly rectifying Cl- conductance via a conventional protein kinase C (PKC). This study examines whether Na+-alanine cotransport similarly activates a DIDS-sensitive Cl- conductance in frog renal proximal tubule cells. On stimulation of Na+-alanine cotransport, the DIDS-sensitive current (I(DIDS-Ala)) increased markedly over time. I(DIDS-Ala) exhibited outward rectification, a Na+/Cl- selectivity ratio of 0.19 +/- 0.03, and an anion selectivity sequence Br- = Cl- > I- > gluconate-. Activation of I(DIDS-Ala) was dependent on ATP hydrolysis and PKC-mediated phosphorylation and was inhibited by hyperosmotic conditions. Activation could be not ascribed to a conventional PKC isoform, as I(DIDS-Ala) was not affected by removing Ca2+ or by phorbol ester treatment, suggesting a role for a nonconventional PKC isoform, either novel or atypical. We conclude that Na+-alanine cotransport activates a DIDS-sensitive Cl- conductance via a nonconventional PKC isoform. This contrasts with the hyposmotically activated Cl- conductance, which requires conventional PKC activation.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Solid variant of alveolar rhabdomyosarcoma with unbalanced t(2;13) and hypotetraploidy, without MYCN amplification.
The histological subtype of alveolar rhabdomyosarcoma (AR) is characterised by the cytogenetic translocation t(2;13)(q35;q14) in approximately 70% of cases, a rearrangement rarely present in the embryonal rhabdomyosarcoma (ER) subtype. The MYCN gene is amplified in some cases of AR. We present a young man with an unusual pattern, namely solid variant of AR with hypotetraploidy and the t(2;13) in an unbalanced form. The MYCN gene was not amplified on FISH, but showed increased copy number, consistent with ploidy.
Adult↗
Forearm blood flow and local responses to peptide vasodilators: a novel pharmacodynamic measure in the phase I trial of antagonist G, a neuropeptide growth factor antagonist.
PURPOSE: Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH(2) (Antagonist G), a substance P (SP 6-11) analogue, inhibits mitogenesis stimulated by a broad spectrum of neuropeptides and has demonstrated antitumor activity in vitro and in vivo with IC(50) concentrations of 10-20 microM in small cell lung cancer and other cell lines. Because neuropeptides are part of complex neurohumoral pathways, we have sought to develop novel pharmacodynamic approaches as part of the early clinical development of this potential anticancer drug. EXPERIMENTAL DESIGN: A Phase I trial was performed in two stages. In stage 1, Antagonist G was administered at 3- week intervals using an accelerated dose-escalation strategy until the target maximum plasma concentration (C(max)) of 10 microM was achieved. In stage 2, dose intensity was increased to weekly, and the inhibitory effect of i.v. Antagonist G was assessed by forearm blood flow (FBF) using SP as a vasodilator, as measured by venous plethysmography. RESULTS: In stage 1, dose was escalated from 2 to 300 mg/m(2) in 12 dose levels using only 15 patients. In stage 2, nine patients were entered at three dose levels (300, 350, and 400 mg/m(2)) and a C(max) of 45 microM was achieved. Facial flushing was the only consistent toxicity but was not dose limiting. FBF studies demonstrated that Antagonist G consistently inhibited the vasodilatory effects of SP (mean, 62 +/- 2% inhibition). CONCLUSIONS: Antagonist G can be safely administered up to 400 mg/m(2), achieving C(max)s >20 microM by weekly 6-h i.v. infusion. FBF studies in patients demonstrated that Antagonist G inhibits SP vasodilatory effects in vivo at these doses in the absence of dose-limiting toxicity.
Adult↗
Stable, polarised, functional expression of Kir1.1b channel protein in Madin-Darby canine kidney cell line.
1. The family of Kir1.1 (ROMK) channel proteins constitute a secretory pathway for potassium in principal cells of cortical collecting duct and thick ascending limb of Henle's loop. Mutations in Kir1.1 account for some types of Bartter's syndrome. 2. Here we report that stable transfection of Kir1.1b (ROMK2) in Madin-Darby canine kidney (MDCK) cell line results in expression of inwardly rectifying K+ currents and transmonolayer electrical and transport properties appropriate to Kir1.1 function. When grown on permeable supports, transfected monolayers secreted K+ into the apical solution. This secretion was inhibited by application of barium to the apical membrane, or by reduction in expression temperature from 37 to 26 C. However, whole-cell voltage clamp electrophysiology showed that K+ conductance was higher in cells expressing Kir1.1b at 26C. 3. To investigate this further, Kir1.1b was tagged with (EGFP), a modification that did not affect channel activity. Protein synthesis was inhibited with cycloheximide. Spectrofluorimetry was used to compare protein degradation at 37 and 26 C. The increased level of Kir1.1b at the plasma membrane at 26 C was due to an increase in protein stability. 4. Confocal microscopic investigation of EGFP-Kir1. 1b fluorescence in transfected cells showed that the channel protein was targeted to the apical domain of the cell. 5. These results demonstrate that Kir1.1b is capable of appropriate trafficking and function in MDCK cell lines at physiological temperatures. In addition, expression of Kir1.1b in MDCK cell lines provides a useful and convenient tool for the study of functional activity and targeting of secretory K+ channels.
Animals↗
The relative risks and etiologic fractions of different causes of death and disease attributable to alcohol, tobacco and illicit drug use in Canada.
BACKGROUND: In 1996 the number of deaths and admissions to hospital in Canada that could be attributed to the use of alcohol, tobacco and illicit drugs were estimated from 1992 data. In this paper we update these estimates to the year 1995. METHODS: On the basis of pooled estimates of relative risk, etiologic fractions were calculated by age, sex and province for 90 causes of disease or death attributable to alcohol, tobacco or illicit drugs; the etiologic fractions were then applied to national mortality and morbidity data for 1995 to estimate the number of deaths and admissions to hospital attributable to substance abuse. RESULTS: In 1995, 6507 deaths and 82,014 admissions to hospital were attributed to alcohol, 34,728 deaths and 194,072 admissions to hospital were attributed to tobacco, and 805 deaths and 6940 admissions to hospital were due to illicit drugs. INTERPRETATION: The use and misuse of alcohol, tobacco and illicit drugs accounted for 20.0% of deaths, 22.2% of years of potential life lost and 9.4% of admissions to hospital in Canada in 1995.
Alcohol Drinking↗
Mosaic rearrangement of chromosome 18: characterization by FISH mapping and DNA studies shows trisomy 18p and monosomy 18p both of paternal origin.
Structural abnormalities of chromosome 18p mainly consist of isochromosomes of the short arm, which result in tetrasomy 18p. Trisomy 18p is much rarer, and less well characterized. We report on a 12-year-old girl with minor facial anomalies, delayed development, abnormal hands, atopic dermatitis, and hearing loss. She was mosaic for two abnormal cell lines in peripheral blood. In 90% of cells, a dicentric chromosome with duplication of the whole short arm of chromosome 18 resulted in trisomy 18p; 10% of cells had monosomy 18p, arising from a t(14;18)(p11;q11). FISH mapping, with multiple region specific and locus specific probes from the short and long arm of chromosome 18, showed that the structure of the dicentric chromosome 18 was 18pter-->18q23::18q11-->18pter. DNA polymorphisms for chromosome 18 showed that the abnormalities of chromosome 18 were paternal in origin. Combining all results, we could link the trisomy 18p and monosomy 18p to a common origin via a complex series of events in an early mitosis.
Abnormalities, Multiple↗
17p- syndrome arising from a novel dicentric translocation in a patient with acute myeloid leukemia.
The cytogenetic contribution to the poor prognosis when myelodysplastic syndrome (MDS) progresses to acute myeloid leukemia (AML) is not well understood. We present a 66-year-old male who had thrombocytopenia with dysplastic features in peripheral blood neutrophils (hypogranular, hyposegmented neutrophils) comprising the Pelger-Huet anomaly, increased blasts in the marrow, and markers consistent with AML. Diagnostic marrow cytogenetics showed a complex karyotype including del(5q), a novel unbalanced dicentric translocation, t(17;20), resulting in both del(20q) and del(17p). Fluorescence in situ hybridization (with probe TP53) showed deletion of 17p13 on the dicentric chromosome, completing the criteria for the 17p- syndrome. Fluorescence in situ hybridization with probes for two tumor suppressor genes on chromosome 5q also showed deletion (CSF1R [at 5(q33.2-q33.4) and EGR-1 [5(q31-q32)]). Remission was difficult to achieve and cytogenetic relapse occurred 6 months postdiagnosis, and clinical relapse approximately one month later. Our case provides a novel mechanism for the 17p- syndrome, and highlights the difficulty of attributing prognostic significance to a particular cytogenetic abnormality in AML.
Aged↗
An intracellular ATP-activated, calcium-permeable conductance on the basolateral membrane of single renal proximal tubule cells isolated from Rana temporaria.
1. The following study describes the properties of a non-selective cation channel, which has a unit conductance below the resolving power of the single channel technique, located on the basolateral membrane of single proximal tubule cells isolated from frog kidney. The conductance was examined using cell-attached, inside-out and outside-out patches. Due to the small single channel magnitude, macroscopic patch currents were measured. 2. Addition of 2 mM ATP to the intracellular surface of excised patches activated an outwardly rectifying conductance (MCANS): outward (Gout) and inward (Gin) conductances increased by 46.8 +/- 6.7 and 11.6 +/- 2.1 pS, respectively (n = 29). MCANS was more selective for cations than anions, with a cation:anion selectivity ratio of 10.1 +/- 1.7 (n = 7), but did not discriminate between Na+ and K+. It was more selective for Ca2+ over Na+, with a Ca2+:Na+ selectivity ratio of 4. 49 +/- 0.69 (n = 7). 3. In cell-attached patches addition of 100 microM strophanthidin to the bath increased both Gout and Gin. However this increase in conductance was absent in the presence of Gd3+, which inhibits MCANS. 4. These data suggest that single proximal tubule cells isolated from frog kidney contain an ATP-activated, non-selective cation conductance. The conductance does not discriminate between Na+ and K+, but is more selective for Ca2+ over Na+. Considering the prevailing electrochemical gradients for these ions, functional activation of the conductance would be expected to lead to a rise in intracellular Ca2+. MCANS is linked to the activity of the Na+, K+-ATPase and may therefore provide a link between the ATPase and K+ channel activity in the basolateral membrane and form an integral part of the pump-leak mechanism in transporting epithelia.
Adenosine Triphosphate↗
Cloning and expression of the Wnt antagonists Sfrp-2 and Frzb during chick development.
The Wnt genes are known to play fundamental roles during patterning and development of a number of embryonic structures. Receptors for Wnts are members of the Frizzled family of proteins containing a cysteine-rich domain (CRD) that binds the Wnt protein. Recently several secreted frizzled-related proteins (Sfrps) that also contain a CRD have been identified and some of these can both bind and antagonise Wnt proteins. In this paper we report the expression patterns of the chick homologues of Frzb, a known Wnt antagonist, and Sfrp-2. Both genes are expressed in areas where Wnts are known to play a role in development, including the neural tube, myotome, cartilage, and sites of epithelial-mesenchymal interactions. Initially, Sfrp-2 and Frzb are expressed in overlapping areas in the neural plate and neural tube, whereas later, they have distinct patterns. In particular Sfrp-2 is associated with myogenesis while Frzb is associated with chondrogenesis, suggesting that they play different roles during development. Finally, we have used the early Xenopus embryo as an in vivo assay to show that Sfrp-2, like Frzb, is a Wnt antagonist. These results suggest that Sfrp-2 and Frzb may function in the developing embryo by modulating Wnt signalling.
Amino Acid Sequence↗
Recombinant anti-carcinoembryonic antigen antibodies for targeting cancer.
Antibodies can be used to target cancer therapies to malignant tissue; the approach is attractive because conventional treatments such as chemo- and radiotherapy are dose limited due to toxicity in normal tissues. Effective targeting relies on appropriate pharmacokinetics of antibody-based therapeutics, ideally showing maximum uptake and retention in tumor and rapid clearance from normal tissue. We have studied the factors influencing these dynamics for antibodies against carcinoembryonic antigen (CEA). Protein engineering of anti-CEA antibodies, in vivo biodistribution models, and mathematical models have been employed to improve understanding of targeting parameters, define optimal characteristics for the antibody-based molecules employed, and develop new therapies for the clinic. Engineering antibodies to obtain the desired therapeutic characteristics is most readily achieved using recombinant antibody technology, and we have taken the approach of immunizing mice to provide high-affinity anti-CEA single-chain Fv antibodies (sFvs) from filamentous bacteriophage libraries. MFE-23, the most characterized of these sFvs, has been expressed in bacteria and purified in our laboratory for two clinical trials: a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumor deposits are detected by a hand-held probe during surgery. Both these trials showed that MFE-23 is safe and effective in localizing tumor deposits in patients with cancer. We are now developing fusion proteins that use the MFE-23 antibody to deliver a therapeutic moiety; MFE-23:: carboxypeptidase G2 (CPG2) targets the enzyme CPG2 for use in the antibody-directed enzyme prodrug therapy system and MFE::tumor necrosis factor alpha (TNFalpha) aims to reduce sequestration and increase tumor concentrations of systemically administered TNFalpha.
Animals↗
A novel dicentric deleted chromosome 21 arising from tandem translocation.
We present a 26-year-old patient with myelodysplastic syndrome (MDS). Initial bone marrow cytogenetics with G-banding showed a rearranged chromosome 21, which was dicentric and bisatellited on CBG- and NOR-banding. Fluorescence in situ hybridization helped to characterize the structure, using a whole chromosome 21 paint and the locus specific AML1 gene probe. The rearranged 21 consisted solely of chromosome 21 material, contained only one copy of AML1, and was not a trisomy, but a deleted tandem translocation. The MDS transformed to acute myeloid leukemia (AML), and the patient died almost 12 months post-diagnosis. Cytogenetics was performed three times during the course of the disease, and the dicentric chromosome 21 was present throughout. Although there are a number of published rearrangements of chromosome 21 in MDS and AML, most are isodicentrics. We could not find another case of an abnormal chromosome 21 with the same structure as reported here.
Acute Disease↗
Appendiceal carcinoma complicating adenomatous polyposis in a young woman with a de novo constitutional reciprocal translocation t(5;8)(q22;p23.1)
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Adenomatous Polyposis Coli↗
Clinical applications of phage-derived sFvs and sFv fusion proteins.
Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNF alpha aims to reduce sequestration and increase tumor concentrations of systemically administered TNF alpha.
Clinical Trials as Topic↗
Low frequency of inherited deletions of 22q11.
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Chromosome Deletion↗
Syndromal obesity due to paternal duplication 6(q24.3-q27).
The likelihood of a paternally expressing imprinted gene in chromosome region 6(q23-24) has been highlighted by cases of transient neonatal diabetes mellitus (TNDM) in which paternal uniparental disomy (UPD) for chromosome 6 or paternal duplication 6(q23-qter) was detected. We present the case of a 38-year-old man with moderate to severe intellectual delay, short stature, small hands and feet, eye abnormality, small mouth, and obesity (without hyperphagia) beginning in mid-childhood. The perinatal and neonatal histories were normal. The patient had a duplication within 6q. Fluorescence in situ hybrisation studies were performed with single and dual hybridisations using a chromosome 6 library probe, short and long arm subregional probes, 6q23-24, 6q25.3-6qter locus-specific probes, and a 6q telomere probe. The hybridisation results defined an inverted duplication of 6q24.3 to 6q27. DNA studies with microsatellite markers from 6p and 6q showed regular biparental inheritance of chromosome 6 and confirmed that the duplication was paternal in origin. Our patient appears to be the first one known to have paternal duplication of chromosome area 6(q24-q27) who did not have TNDM as an infant. He has remained nondiabetic, although obesity, without hyperphagia, has been a constant problem since its onset in mid-childhood.
Adult↗
Stimulation of Na+-alanine cotransport activates a voltage-dependent conductance in single proximal tubule cells isolated from frog kidney.
1. The swelling induced by Na+-alanine cotransport in proximal tubule cells of the frog kidney is followed by regulatory volume decrease (RVD). This RVD is inhibited by gadolinium (Gd3+), an inhibitor of stretch-activated channels, but is independent of extracellular Ca2+. 2. In this study, the whole cell patch clamp technique was utilized to examine the effect of Na+-alanine cotransport on two previously identified volume- and Gd3+-sensitive conductances. One conductance is voltage dependent and anion selective (GVD) whilst the other is voltage independent and cation selective (GVI). 3. Addition of 5 mM L-alanine to the bathing solution increased the whole cell conductance and gave a positive (depolarizing) shift in the reversal potential (Vrev, equivalent to the membrane potential in current-clamped cells) consistent with activation of Na+-alanine cotransport. Vrev shifted from -36 +/- 4.9 to +12.9 +/- 4.2 mV (n = 15). 4. In the presence of alanine, the total whole cell conductance had several components including the cotransporter conductance and GVD and GVI. These conductances were separated using Gd3+, which inhibits both GVD and GVI, and the time dependency of GVD. Of these two volume-sensitive conductances, L-alanine elicited a specific increase in GVD, whereas GVI was unaffected. 5. The L-alanine-induced activation of GVD was significantly reduced when cells were incubated in a hypertonic bathing solution. 6. In summary, in single proximal tubule cells isolated from frog kidney, on stimulation of Na+-alanine cotransport GVD is activated, while GVI is unaffected. Taken with other evidence, this suggests that GVD is activated by cell swelling, consequent upon alanine entry, and may play a role as an anion efflux pathway during alanine-induced volume regulation.
Alanine↗