William G. Tsiaras

Receptor-Specific Regulation of Phosphatidylinositol 3′-Kinase Activation by the Protein Tyrosine Phosphatase Shp2

ABSTRACT Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3′-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.

Factors Influencing Vitamin D Status

The steroid hormone vitamin D is required for normal calcium and phosphorus metabolism and is thus an important contributor to musculoskeletal health. Recent data have linked low vitamin D levels to a wide range of diseases, including cancer, cardiovascular disease, autoimmune disease and infection. Adequate levels of vitamin D are maintained through its cutaneous photosynthesis and oral ingestion. By some estimates, one billion people worldwide have vitamin D deficiency or insufficiency. A number of factors influence the photosynthesis and bioavailability of vitamin D and contribute to risk of impaired vitamin D status. These factors include variation in sun exposure due to latitude, season, time of day, atmospheric components, clothing, sunscreen use and skin pigmentation, as well as age, obesity and the incidence of several chronic illnesses. This review will focus on factors that influence vitamin D status and contribute to the prevalence of low vitamin D levels.

Mutations in the GIGYF2 (TNRC15) Gene at the PARK11 Locus in Familial Parkinson Disease

The genetic basis for association of the PARK11 region of chromosome 2 with familial Parkinson disease (PD) is unknown. This study examined the GIGYF2 (Grb10-Interacting GYF Protein-2) (TNRC15) gene, which contains the PARK11 microsatellite marker with the highest linkage score (D2S206, LOD 5.14). The 27 coding exons of the GIGYF2 gene were sequenced in 123 Italian and 126 French patients with familial PD, plus 131 Italian and 96 French controls. A total of seven different GIGYF2 missense mutations resulting in single amino acid substitutions were present in 12 unrelated PD index patients (4.8%) and not in controls. Three amino acid insertions or deletions were found in four other index patients and absent in controls. Specific exon sequencing showed that these ten sequence changes were absent from a further 91 controls. In four families with amino acid substitutions in which at least one other PD case was available, the GIGYF2 mutations (Asn56Ser, Thr112Ala, and Asp606Glu) segregated with PD. There were, however, two unaffected carriers in one family, suggesting age-dependent or incomplete penetrance. One index case (PD onset age 33) inherited a GIGYF2 mutation (Ile278Val) from her affected father (PD onset age 66) and a previously described PD-linked mutation in the LRRK2 gene (Ile1371Val) from her affected mother (PD onset age 61). The earlier onset and severe clinical course in the index patient suggest additive effects of the GIGYF2 and LRRK2 mutations. These data strongly support GIGYF2 as a PARK11 gene with a causal role in familial PD.

Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs

GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3), with KD values of 0.05, 1.6 and 1.0 μm for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4,5)P3 versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly274-Gly275 motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P2 with little effect on its binding to PtdIns(3,4,5)P3, while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P2 but not for PtdIns(3,4,5)P3. In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P3 binding selectivity.

Prognostic importance of ophthalmic manifestations in childhood leukaemia.

In order to assess the systemic prognosis of children with leukaemic ocular involvement, 63 of 131 patients admitted to hospital with acute leukaemia were evaluated ophthalmically. A total of 28 of 63 showed ophthalmic involvement and were followed up for up to 84 months. Twenty seven of 28 patients (96.4%) died within 28 months after the onset of ocular involvement and within 83 months after the onset of leukaemia. The 5 year survival rate of patients with ophthalmic manifestations was 21.4% (6/28). This survival rate was significantly lower than that of those who lacked ophthalmic manifestations (16/35: 45.7%, p < 0.05). All of the patients with ophthalmic manifestations had either bone marrow relapse or central nervous system leukaemia. The prognosis was related to risk factors such as central nervous system leukaemia or bone marrow relapse in most cases.

Amblyopia and visual acuity in children with Down’s syndrome

BACKGROUND/AIMS Amblyopia in people with Down’s syndrome has not been well investigated. This study was designed to determine the prevalence and associated conditions of amblyopia in a group of home reared children with Down’s syndrome. METHODS All children in the study group underwent an evaluation of visual acuity. In addition, previous ophthalmological records were reviewed, and a subgroup of children was examined. For the purposes of this study, amblyopia was defined quantitatively as a difference of two Snellen acuity lines between eyes or if unilateral central steady maintained (CSM) vision and a clear fixation preference was observed. A high refractive error was defined as a spherical equivalent more than 3 dioptres and astigmatism more than 1.75 dioptres. Anisometropia was defined as a difference of at least 1.5 dioptres of sphere and/or 1.0 dioptre of cylinder between eyes. 68 children with Down’s syndrome between the ages of 5 and 19 years were enrolled in the final study group. RESULTS Amblyopia was observed in 15 (22%) of 68 patients. An additional 16 (24%) patients had bilateral vision less than 20/50. Strabismus, high refractive errors, and anisometropia were the conditions most commonly associated with decreased vision and amblyopia CONCLUSION This study suggests that the prevalence of amblyopia is higher than previously reported. Fully 46% of these children with Down’s syndrome had evidence of substantial visual deficits. These patients may be at higher risk for visual impairment and should be carefully examined for ophthalmological problems.

Intralesional Sodium Thiosulfate for the Treatment of Calciphylaxis

IMPORTANCE Calciphylaxis is a potentially fatal disorder of abnormal calcium deposition. Patients commonly present with painful retiform to stellate purpuric lesions that often undergo ulceration and necrosis, increasing the risk of infection and life-threatening sepsis. Treatment is multifaceted, and improved outcomes have been demonstrated with intravenous sodium thiosulfate; however, the use of this medication can be limited by its adverse effects. The use of topical sodium thiosulfate has been successfully reported for superficial calcium deposits in the skin from other processes. Therefore, we hypothesized that intralesional (IL) sodium thiosulfate may be an effective treatment for the deeper lesions of cutaneous calciphylaxis. We provide a retrospective case review of 4 patients with calciphylaxis who were treated with IL sodium thiosulfate. OBSERVATIONS Four patients with biopsy-proven cutaneous calciphylaxis were treated with IL sodium thiosulfate (250 mg/mL) in areas of clinically active disease. The patients tolerated the medication well, with only transient localized discomfort during injection. All 4 patients had complete healing of their ulcers and remission of disease. CONCLUSIONS AND RELEVANCE Intralesional sodium thiosulfate may be an effective and well-tolerated treatment for localized calciphylaxis. This novel approach requires further research and investigation.

IGF-I Activation of the AKT Pathway Is Impaired in Visceral But Not Subcutaneous Preadipocytes from Obese Subjects

Obesity morbidity is associated with excess visceral adiposity, whereas sc adipose tissue is much less metabolically hazardous. Human abdominal sc preadipocytes have greater capacity for proliferation, differentiation, and survival than omental preadipocytes. IGF-I is a critical mediator of preadipocyte proliferation, differentiation, and survival through multiple signaling pathways. We investigated IGF-I action in primary cultures of human preadipocytes isolated from sc and omental adipose tissue of obese subjects. IGF-I-stimulated DNA synthesis was significantly lower in omental compared with sc preadipocytes. IGF-I phosphorylation of the IGF-I receptor and the ERK pathway was comparable in sc and omental cells. However, omental preadipocytes had decreased insulin receptor substrate (IRS)-1 protein associated with increased IRS-1-serine(636/639) phosphorylation and degradation. IGF-I-stimulated phosphorylation of AKT on serine(473) but not threonine(308) was decreased in omental cells, and activation of downstream targets, including S6Kinase, glycogen synthase kinase-3, and Forkhead box O1 was also impaired. CyclinD1 abundance was decreased in omental cells due to increased degradation. Over-expression of IRS-1 by lentivirus in omental preadipocytes increased IGF-I-stimulated AKT-serine(473) phosphorylation. The mammalian target of rapamycin (mTOR)-Rictor complex regulates phosphorylation of AKT-serine(473) in 3T3-L1 adipocytes, but knockdown of Rictor by lentivirus-delivered short hairpin RNA in sc preadipocytes did not affect AKT-serine(473) phosphorylation by IGF-I. These data reveal an intrinsic defect in IGF-I activation of the AKT pathway in omental preadipocytes from obese subjects that involves IRS-1 but probably not mTOR-Rictor complex. We conclude that impaired cell cycle regulation by AKT contributes to the distinct growth phenotype of preadipocytes in visceral fat of obese subjects.

GIGYF2 Gene Disruption in Mice Results in Neurodegeneration and Altered Insulin-like Growth Factor Signaling

Grb10-Interacting GYF Protein 2 (GIGYF2) was initially identified through its interaction with Grb10, an adapter protein that binds activated IGF-I and insulin receptors. The GIGYF2 gene maps to human chromosome 2q37 within a region linked to familial Parkinsons disease (PARK11 locus), and association of GIGYF2 mutations with Parkinsons disease has been described in some but not other recent publications. This study investigated the consequences of Gigyf2 gene disruption in mice. Gigyf2 null mice undergo apparently normal embryonic development, but fail to feed and die within the first 2 post-natal days. Heterozygous Gigyf2(+/-) mice survive to adulthood with no evident metabolic or growth defects. At 12-15 months of age, the Gigyf2(+/-) mice begin to exhibit motor dysfunction manifested as decreased balance time on a rotating horizontal rod. This is associated with histopathological evidence of neurodegeneration and rare intracytoplasmic Lewy body-like inclusions in spinal anterior horn motor neurons. There are alpha-synuclein positive neuritic plaques in the brainstem and cerebellum, but no abnormalities in the substantia nigra. Primary cultured embryo fibroblasts from Gigyf2 null mice exhibit decreased IGF-I-stimulated IGF-I receptor tyrosine phosphorylation and augmented ERK1/2 phosphorylation. These data provide further evidence for an important role of GIGYF2 in age-related neurodegeneration and IGF pathway signaling.

Effect of lateral cervical cord lesions on the respiratory rhythm of anaesthetized, decerebrate cats after vagotomy

1. The lateral cervical cord of vagotomized, anaesthetized cats was superficially lesioned at the C3 or the C7 level before or after midcollicular decerebration.