Transfection of primary human skin fibroblasts by electroporation

Primary human skin fibroblasts are an accessible source of phenotypically and karyotypically normal human cells, but are difficult to transfect with exogenous DNA. Here we demonstrate that both transient expression and stable transformation can be carried out by the method of electroporation. Highly efficient transient chloramphenicol acetyltransferase expression was shown after transfection with plasmid pRSVCAT. Stable transformation of human skin fibroblasts to G418 resistance was obtained after electroporation with neo-containing plasmids at an efficiency of approximately 1.4 x 10-5/[mu]g DNA. The ability to easily transfect these cells with exogenous DNA may have important applications in the study of human genetic diseases and cancer.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27179/1/0000177.pd

The Concise Guide to PHARMACOLOGY 2023/24: Ion channels

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

Loss of the p16INK4a and p15INK4b genes, as well as neighboring 9p21 markers, in sporadic melanoma

Although homozygous deletions of the cyclin-dependent kinase inhibitor 2 gene p16(INK4a) on 9p21 have been reported frequently in metastatic melanoma cell lines, and intragenic mutations within the p16(INK4a) gene have been detected in familial melanoma kindreds, specific targeting of this gene in the development of sporadic melanoma in vivo remains controversial. Southern analyses were performed in this study to initially assess the frequency of hemi- or homozygous losses of p16(INK4a), as well as its neighboring family member, p15(INK4b), and other candidate regions within 9p21, in sporadic melanoma. Overall, 22 of 40 (55%) uncultured sporadic melanoma DNAs were determined in harbor deletions of 1-11 markers/genes located on 9p21. This included 10 tumors (25%; 10 of 40) with homozygous deletions limited to either the p16(INK4a) gene only (20%; 2 of 10), both the p16(INK4a) and p15(INK4b) genes (10%; 1 of 10), another novel 9p21 gene, FB19 (10%; 1 of 10), or all three of these genes plus surrounding markers (60%; 6 of 10). In subsequent single-strand conformation polymorphism and sequencing analyses, intragenic mutations in the p16(INK4a) gene were also revealed in two (10%; 2 of 21) melanoma DNAs that retained one copy of this locus. By comparison, the frequency of p16(INK4a) and p15(INK4b) homozygous deletions, as well as p16(INK4a) mutations, in melanoma cell lines (analyzed in parallel) was 2-3- fold higher at 61 (23 of 38) and 24% (9 of 38), respectively. These findings indicate that (a) p16(INK4a) is inactivated in vivo in over one-fourth (27.5%; 11 of 40) of sporadic melanomas; (b) mutation/deletion of p16(INK4a) may confer a selective growth advantage in vitro; and (c) other 9p21 tumor suppressor genes could be targeted during the development of melanoma.This work was supported in part by the National Health and Medical Research Council of Australia.Peer Reviewe

MspI RFLP detected by a ZNF-40 cDNA sequence

Source/Description: 1.6 kb EcoRI fragment from a 5' cDNA clone (designated 4b) of the human ZNF-40 locus. ZNF-40 (MBP-1) (PRDII-BFI) encodes the class I major histocompatibility complex (MHC) enhancer binding protein 1 (2)

Analysis of the CDKN2A, CDKN2B and CDK4 genes in 48 Australian melanoma kindreds

Germline mutations within the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and one of its targets, the cyclin dependent kinase 4 (CDK4) gene, have been identified in a proportion of melanoma kindreds. In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma. We have previously reported the identification of germline CDKN2A mutations in 7/18 Australian melanoma kindreds and the absence of the R24C CDK4 mutation in 21 families lacking evidence of a CDKN2A mutation. The current study represents an expansion of these efforts and includes a total of 48 melanoma families from Australia. All of these families have now been screened for mutations within CDKN2A and CDK4, as well as for mutations within the CDKN2A homolog and 9p21 neighbor, the CDKN2B gene, and the alternative exon 1 (E1beta) of CDKN2A. Families lacking CDKN2A mutations, but positive for a polymorphism(s) within this gene, were further evaluated to determine if their disease was associated with transcriptional silencing of one CDKN2A allele. Overall, CDKN2A mutations were detected in 3/30 (10%) of the new kindreds. Two of these mutations have been observed previously: a 24 bp duplication at the 5' end of the gene and a G to C transversion in exon 2 resulting in an M531 substitution. A novel G to A transition in exon 2, resulting in a D108N substitution was also detected. Combined with our previous findings, we have now detected germline CDKN2A mutations in 10/48 (21%) of our melanoma kindreds. In none of the 'CDKN2A-negative' families was melanoma found to segregate with either an untranscribed CDKN2A allele, an R24C CDK4 mutation, a CDKN2B mutation, or an E1beta mutation. The last three observations suggest that these other cell cycle control genes (or alternative gene products) are either not involved at all, or to any great extent, in melanoma predisposition