The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis

Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disease that affects the aorta, carotid, coronary and pulmonary arteries. Previous molecular genetic data have led to the hypothesis that SVAS results from mutations in the elastin gene, ELN. In these studies, the disease phenotype was linked to gross DNA rearrangements (35 and 85 kb deletions and a translocation) in three SVAS families. However, gross rearrangements of ELN have not been identified in most cases of autosomal dominant SVAS. To define the spectrum of ELN mutations responsible for this disorder, we refined the genomic structure of human ELN and used this information in mutational analyses. ELN point mutations co-segregate with the disease in four familial cases and are associated with SVAS in three sporadic cases. Two of the mutations are nonsense, one is a single base pair deletion and four are splice site mutations. In one sporadic case, the mutation arose de novo. These data demonstrate that point mutations of ELN cause autosomal dominant SVAS.      

Immunological studies of the glycoprotein allergen Ag-54 (Cla h II) in Cladosporium herbarum with special attention to the carbohydrate and protein moieties

The glycoprotein allergen Ag-54 (Cla h II) isolated from Cladosporium herbarum was split into its carbohydrate and protein moieties by using alkaline-borohydride treatment and deglycosylation, respectively. The native Ag-54, the deglycosylated protein and the protein-free carbohydrate moieties were tested for IgE-binding activity by radioallergosorbent test inhibition using selected sera from individuals with C. herbarum allergy. The deglycosylated material had a stronger allergenic activity than the native Ag-54 with all the sera tested. The Ag-54 carbohydrate moieties were not found to possess any IgE-binding activity. The galactoglucomannan part of the carbohydrate moiety was isolated and it was demonstrated to precipitate IgG rabbit antibody. Removal of the galactofuranose units by mild acid hydrolysis did not alter the IgG-binding capacity of the polysaccharide, indicating that galactofuranose was not immunodominant.     

The E3-11.6K protein of adenovirus is an Asn-glycosylated integral membrane protein that localizes to the nuclear membrane.

The 11,600 MW (101 amino acids; 11.6K) protein of adenovirus 2 (Ad2) is a protein of unknown function which is synthesized in low amounts during early stages of infection but in very high amounts at late stages. The 11.6K protein migrates as three major groupings of diffuse bands of ca. 14K, 21K, and 31K on SDS-PAGE, indicating that 11.6K undergoes post-translational modification. We show here that 11.6K is Asn-glycosylated with complex (endo H-resistant) oligosaccharides and that 11.6K is an integral membrane protein. Immunofluorescence indicated that 11.6K initially is associated with the endoplasmic reticulum and Golgi apparatus and that it ultimately localizes to the nuclear membrane. The 11.6K protein is predicted to have a single signal-anchor sequence at residues 41-62 and only one potential Asn-linked glycosylation site at residue 14; thus, 11.6K must be oriented in the membranes with its NH2-terminus in the lumen and its COOH-terminus in the cytoplasm. The signal-anchor and glycosylation features of 11.6K are preserved in Ad2 and Ad5 (group C), and in Ad3 and Ad7 (group B), but the sequence of 11.6K is more diverged among these serotypes than is the sequence of most other adenovirus proteins.     

Identification of families of overlapping polypeptides coded by early "transforming" gene region 1 of human adenovirus type 2.

Antisera against four lines of adenovirus 2-transformed rat cells, including F17 cells which contain only early gene region 1 (map position 1.5–11) (the transforming region), immunoprecipitate major polypeptides of 53,000 (53K) and 15,000 (15K) daltons (Gilead, Y.-H. Jeng, W. S. M. Wold, K. Sugawara, H. M. Rho, M. L. Harter, and M. Green, 1976, Nature (London)264,263–266; W. S. M. Wold and M. Green, 1976, J. Virol.30,297–310). We show here that the 53Ks precipitated by three of these antisera as well as Adl-SV40 induced hamster tumor sera all have identical two-dimensional tryptic [³⁵S]Met-peptide maps. Similarly, all 15Ks precipitated by these antisera have identical maps. Tryptic and chymotryptic maps demonstrate that most if not all of the [³⁵S]Met-peptides of 15K are shared by 53K. Recent tryptic peptide maps (D. Halbert, D. Spector, H. J. Raskas, W. S. M. Wold, and M. Green, unpublished data) prove that the 53K corresponds to a 53K translated in vitro from a 22S RNA derived from map positions 4.5–11 (D. Halbert, D. Spector, and H. J. Raskas, J. Virol., in press). Thus, both the 53K and 15K are coded within map position 4.5–11. Four [³⁵S]Met-labeled polypeptides of 40K–50K, specific to Ad2 early infected human cells, were isolated in O'Farrell-type 2D gels. These polypeptides are coded within map position 1.5-4.5 (D. Halbert, D. Spector, and H. J. Raskas, J. Virol., in press; D. Halbert, D. Spector, H. J. Raskas, W. S. M. Wold, and M. Green, unpublished data). As expected, our tryptic and chymotryptic peptide maps of these four polypeptides indicate no relationship to 53K or 15K. Two of the 40K–50K polypeptides are highly related, the other two are highly related, and all four are partially related. In addition to 53K and 15K, the F17 antiserum precipitated a 28K polypeptide and minor bands of 14K–16K, 18K–20K, and 11K–12K. Tryptic and chymotryptic maps of these polypeptides show that (1)14K–16K and 18K–20K polypeptides are members of the 53K/15K family, (2) 11K–12K polypeptides are related to each other but are unrelated to any other polypeptides, and (3) 28K is unrelated to any other polypeptides. We conclude that early region 1 codes for two (53K/15K family, 40K–50K family) or possibly more (e.g., 11K–12K family) families of polypeptides; the polypeptides included in each family overlap in amino acid sequence and therefore may be translated from overlapping spliced mRNAs. The possible contribution of these polypeptides to the phenotype of Ad2-transformed cells is discussed.     

Short-term therapy for recurrent abortion using intravenous immunoglobulins: results of a double-blind placebo-controlled Italian study

It is still unclear whether i.v. immunoglobulins (Ig) can facilitate the reproductive prognosis of women who have suffered recurrent pregnancy loss. We report the results of a multicentre placebo- controlled study on the effect of Ig administration on pregnancy outcome in 46 women who had suffered at least three recurrent miscarriages. All were screened to exclude chromosomal or Mullerian abnormalities, the presence of antinuclear antibodies, lupus anticoagulant (LA) or elevated titres of anticardiolipin antibodies which may have revealed an underlying autoimmune problem. To avoid a selection bias towards ongoing pregnancies, i.v. Ig or placebo were administered between weeks 5 and 7 of gestation for 2 consecutive days as soon as each woman knew she was pregnant and before embryonic heart activity could be detected. A further infusion was administered at week 8 when ultrasonography confirmed an ongoing embryonic development. In all, 68% of the women who received Ig went to term versus 79% of those who received a placebo (not significant), with no significant differences in the pregnancy course or the perinatal outcome. These results suggest either that women with recurrent miscarriages who have no recognized cause of pregnancy loss have a good reproductive prognosis without any treatment or that the emotional care associated with the administration of a placebo can indirectly facilitate the progression of pregnancy.