Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart

Myotonic dystrophy (DM) is a multisystemic disorder caused by an inherited CTG repeat expansion which affects three genes encoding the DM protein kinase (DMPK), a homeobox protein Six5 and a protein containing WD repeats. Using a panel of 16 monoclonal antibodies against several different DMPK epitopes we detected DMPK, as a single protein of approximately 80 kDa, only in skeletal muscle, cardiac muscle and, to a lesser extent, smooth muscle. Many earlier reports of DMPK with different sizes and tissue distributions appear to be due to antibody cross-reactions with more abundant proteins. One such antibody, MANDM1, was used to isolate two related protein kinases, MRCK alpha and beta, from a human brain cDNA library and the shared epitope was located at the catalytic site of DMPK using a phage-displayed random peptide library. The peptide library also identified an epitope shared between DMPK and a 55 kDa muscle-specific protein. The results suggest that effects of the repeat expansion on the DMPK gene may be responsible for muscle and heart features of DM, whereas clinical changes in other tissues may be due to effects on the other two genes.     

Isolation of human single chain antibodies (scFv) against human TNF-alpha from human peripheral blood lymphocyte-SCID mice

By developing an appropriate immunization protocol for SCID (hu-PBL-SCID) mice engrafted with human peripheral blood lymphocytes in combination with scFv phage display library, we were able to establish an efficient strategy to obtain human scFv clones against a human self-antigen, TNF-alpha. The mice pretreated with gamma-radiation (3Gy) and anti-asialo GM1 antibody were immunized with a mixture of human TNF-alpha-keyhole limpet hemocyanin and Freund's adjuvant. Human antibody maturation was suggested to be induced in the mice with the immunization protocol. The scFv clones obtained from the mice were shown to exhibit binding affinities in the range of 10(7)-10(8) M(-1). Together with our previously published work on the isolation of respiratory syncytial virus neutralizing scFvs, the results of this study have implicated that this combined approach is one of the effective alternatives for the cloning of human monoclonal antibodies specific for a wide range of antigens of interest.     

Germline mutations of the CDKN2 gene in UK melanoma families

Germline mutations in CDKN2 on chromosome 9p21, which codes for the cyclin D kinase inhibitor p16, and more rarely, mutations in the gene coding for CDK4, the protein to which p16 binds, underlie susceptibility in some melanoma families. We have sequenced all exons of CDKN2 and analysed the CDK4 gene for mutations in 27 UK families showing evidence of predisposition to melanoma. Five different germline mutations in CDKN2 were found in six families. Three of the mutations (Met53Ile, Arg24Pro and 23ins24) have been reported previously. We have identified two novel CDKN2 mutations (88delG and Ala118Thr) which are likely to be associated with the development of melanoma, because of their co-segregation with the disease and their likely functional effect on the CDKN2 protein. In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma. Ala118Thr appeared to be functional in this assay. Arg24Pro appeared to bind to CDK6, but not to CDK4. No mutations were detected in exon 2 of CDK4, suggesting that causal mutations in this gene are uncommon. The penetrance of these mutant CDKN2 genes is not yet established, nor is the risk of non-melanoma cancer to gene carriers.      

Simian retrovirus vectors for gene transfer in nonhuman primate cells

We have recently identified and sequenced a molecular clone of the serogroup 2 simian retrovirus (SRV), D2/RHE/OR/V1, that retains an enhanced ability to infect specific T cell lines. In this report, using deletion mutagenesis, we localized the psi packaging signal, necessary for packaging of D2/RHE/OR/V1 particles, to the genomic region 345-650, which comprises the 5' intergenic region (IR) and the extreme 5' portion of the gag gene. To build an SRV-based gene transfer system and to reduce the possibility of recombination and regeneration of replication-competent viruses, we constructed split-genome D2/RHE/OR/V1 plasmid recombinants containing distinct and non-overlapping retroviral gene regions and several replacement components. For the retrovirus gene transfer vehicle, we deleted the D2/RHE/OR/V1 structural genes and substituted a cassette including the psi-packaging region, the beta-galactosidase reporter gene, and the 3' IR. Both packaging cell recombinants were used to generate stable monkey packaging cell lines; the gene transfer vehicle was subsequently transfected into the packaging cell lines, and replication-defective viruses were recovered for subsequent infection into fresh monkey cells. Successful infection by the recovered viruses verifies the potential efficacy of the SRV-based system as a research tool for gene transfer of heterologous genes into nonhuman primate cells.     

'Fat mass and obesity associated' gene (<Emphasis Type="Italic">FTO</Emphasis>): No significant association of variant rs9939609 with weight loss in a lifestyle intervention and lipid metabolism markers in German obese children and adolescents

Background  

We have previously identified strong association of six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) to early onset extreme obesity within the first genome wide association study (GWA) for this phenotype. The aim of this study was to investigate whether the obesity risk allele of one of these SNPs (rs9939609) is associated with weight loss in a lifestyle intervention program. Additionally, we tested for association of rs9939609 alleles with fasting blood parameters indicative of glucose and lipid metabolism.     

Comparative study on deletions of the dystrophin gene in three Asian populations

The frequency and distribution of deletions of 19 deletion-prone exons clustered in two hot spots in the proximal and central regions of the dystrophin gene were compared in three populations from Singaporean, Japan, and Vietnam. DNA samples obtained from 105 Singaporean, 86 Japanese, and 34 Vietnamese Duchenne muscular dystrophy patients were examined by polymerase chain reaction amplification. Deletions of the examined exons were found in 51.2% of Japanese patients but in 40.0% or less of the Singaporeans and Vietnamese. About two thirds of the deletions were localized in the central region and the remaining deletions were clustered at the proximal region. The most commonly deleted exons at the central deletion hot spot were exon 50 in the Singaporean, exons 49 and 50 in the Japanese, and exon 51 in the Vietnamese population. At the proximal deletion hot spot, the most commonly deleted exons were exons 6 and 8 in the Singaporeans, exons 12 and 17 in the Japanese, and exons 8 and 12 in the Vietnamese. Two cases each from Singapore and Japan had large-scale gross mutations spanning both deletion hot spots. Our results suggest that, although the presence and frequency of the two deletion hot spots may be similar in the three Asian populations analyzed, the distribution and frequency of deletions among the different exons can vary as a result of population-specific intronic sequences that predispose individuals to preferential deletion breakpoints. Received: May 20, 2002 / Accepted: July 1, 2002     

Multilineage potential of homozygous stem cells derived from metaphase II oocytes

Human stem cells derived from human fertilized oocytes, fetal primordial germ cells, umbilical cord blood, and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However, the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach, employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D), we established stem cell lines homozygous for H-2-B and H-2-D, respectively. The undifferentiated cells retained a normal karyotype, expressed stage-specific embryonic antigen-1 and Oct4, and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition, these cells demonstrated the potential for in vitro differentiation into endoderm, neuronal, and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes, and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible "homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation, while decreasing the ethical concerns surrounding human embryonic stem cell research.     

In vitro activity of cefpodoxime against bacterial isolates obtained from patients with cancer

The in vitro activity of cefpodoxime, an oral cephalosporin ester, against 792 bacterial isolates representing 36 species was evaluated in comparison to that of ciprofloxacin and trimethoprim/sulfamethoxazole (TMP/SMX). Cefpodoxime inhibited the majority ofStreptococcus spp.,Haemophilus influenzae andProteus mirabilis at a concentration of 0.12 µg/ml. It was also active againstCitrobacter diversus, Escherichia coli, Klebsiella spp.,Proteus vulgaris, Serratia marcescens and methicillin-susceptibleStaphylococcus aureus isolates. Overall, cefpodoxime appeared to be less active than ciprofloxacin and TMP/SMX against many pathogens common in cancer patients.