Biochemical and morphological analysis of cell death induced by Egyptian cobra (Naja haje) venom on cultured cells

We investigated the in vitro process of cell death caused by Egyptian cobra venom on primary human embryonic kidney (293T) and mouse myoblast (C2C12) cell lines. The aim of these studies was to provide further information about triggering cell death, and suggest methods for eliminating unwanted cells, such as tumour cells. Both cell lines were treated with 10, 20, and 50 m g/ml of Egyptian cobra (Naja haje) venom in serum free media (SFM) and incubated for 8 hours. Total activities of the lactate dehydrogenase (LDH) and creatine kinase (CK) released in the culture during venom incubation were used as an indicator of the venom in vitro cytotoxicity. Cell injury was morphologically recognized and apoptosis determined by a Fluorescing Apoptosis Detection System and confirmed by staining nuclear DNA with DAPI. Our data clearly demonstrated marked cytotoxic effects and acute cell injury for both cell lines. Release of LDH and CK into the culture media induced by the venom correlates well with the morphological changes and extent of cell death. Mostly, these consequences were time and dose-dependent in both cell lines. The results obtained from this study indicated that cobra venom cause cell death by two different mechanisms: necrosis and induction of apoptosis. The apoptotic mechanism, accompanied by cell necrosis, mediated cell destruction of both tested cell lines; however, necrosis was predominant in the C2C12 cell line while apoptosis, in 293T cells. This unusual form of cell death induced by cobra venom may represent a combination of apoptosis and necrosis within the same cell. This is a first-hand investigation showing the apoptotic effects of N. haje venom at the cellular level. However, the contribution of the apoptotic pathway may be dependent on concentration and/or time of exposure to snake venom.(AU)

Recombinant micro-genes and dystrophin viral vectors.

An effective gene therapy for Duchenne muscular dystrophy ideally relies on the ability to provide long-term expression to muscle tissue of the missing protein, dystrophin. Early work in the mdx mouse using a 6.3 kb mini-dystrophin cDNA, carried out in either adenoviral or retroviral vectors was generally successful, however, expression was only transient. In an attempt to remedy this problem, two approaches are being investigated. The first of these is a hybrid vector system that combines the efficacy of gene transfer into skeletal muscle of adenoviral vectors with the long-term stability of retroviral vectors. The second utilises the inherently efficient transducing properties and stability of the adeno-associated viral delivery system. Using highly truncated micro-dystrophin cDNAs we have shown that both vector systems were able to restore dystrophin and dystrophin-associated protein expression at the plasma membrane of mdx mice for prolonged periods of time. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. These studies suggest that hybrid adenoviral-retroviral and adeno-associated viral vectors are capable of ameliorating dystrophic pathology at the cellular level and as such are useful tools in the development of a gene therapy for Duchenne muscular dystrophy.

Technology evaluation: AAV factor IX gene therapy, Avigen Inc.

Avigen has developed a recombinant adeno-associated viral vector expressing human blood-coagulation Factor IX (F.IX) for the potential treatment of hemophilia B. In a phase I clinical trial being conducted at The Children's Hospital of Philadelphia and Stanford University Medical Center, the vector, AAV-CMV-hF.IX (Coagulin-B), was injected at a low dose into three patients with severe hemophilia B. No evidence of toxicity, germline transmission of vector sequences, or formation of inhibitory antibodies against F.IX was observed, and in two of the three patients there was an indication of a modest clinical response.

Allelic variation of ovine MHC class II DQA1 and DQA2 genes.

In the present study we characterize allelic variation of polymorphic OLA-DQA1 and OLA-DQA2 genes in sheep. To achieve this, PCR primers were designed to independently amplify the second exons of OLA-DQA1 and OLA-DQA2 genes. Single strand conformation polymorphism (SSCP) gel analyses reveals that there are at least 12 distinct OLA-DQA2 sequences, 10 of which have been characterized by sequencing. Six distinct OLA-DQA1 alleles have been sequenced in sheep and we can detect at least seven DQA1 alleles, including a null allele, by SSCP analysis. The second exon of the OLA-DQA2 gene is more polymorphic than the equivalent region of the OLA-DQA1 gene. Thirty-two per cent of nucleotide and 49% of amino acid sites showed variation at the DQA2 locus, compared to 20% of nucleotide and 33% of amino acid sites for DQA1. Phylogenetic analysis of DQA sequences from a number of species show that sheep DQA1 sequences group together and are more similar to bovine DQA1 sequences than to sheep DQA2 alleles. The majority of OLA-DQA2 sequences are on the same main branch of the phylogenetic tree as bovine DQA2 sequences. However, three sheep DQA2 sequences have a tendency to group with putative bovine DQA3 sequences rather than to other ovine DQA2 alleles. A variety of SSCP gel conditions were tried in order to develop a typing system for the OLA-DQA2 gene. We describe a set of PCR and SSCP conditions which distinguish between all known OLA-DQA2 alleles.

Generation of novel human MHC class II mutant B-cell lines by integrating YAC DNA into a cell line homozygously deleted for the MHC class II region.

The human B lymphoblastoid cell line (LCL) 721.174 sustains a large homozygous deletion in the major histocompatibility complex (MHC) class II region that results in an absence of DQ and DR molecules as well as a deficiency in the assembly and transport of class I molecules to the cell surface. The deleted genes include the transporters associated with antigen processing TAP1 and TAP2, DMA and DMB which are involved in editing class II bound peptides, and four genes whose roles in antigen processing are unclear; the low mass polypeptide genes LMP2 and LMP7, and DNA and DOB. To study this region we have integrated into 721.174 two overlapping yeast artificial chromosome (YAC) clones which cover the interval LMP2-DRA inclusive. Three clones (11.2A1.1, 4D1D10.1 and 4D1D10.2), containing complete copies of the transfected YAC, produced varying levels of mRNA from the LMP, TAP, DQ and DR genes and corresponding levels of LMP and TAP protein. Class I cell surface expression was restored in 11.2A1.1 and 4D1D10.1, as was DR expression in both 4D1D10 transfectants. These studies demonstrate the feasibility of introducing large groups of functional genes back into human lymphoblastoid cells sustaining deletions, with full restoration of biological function. The procedure could be exploited in order to restore all but one gene covered by the deletion, effectively producing a single gene defect. This could be used to introduce copies of genes engineered to contain mutations and to study cis regulatory elements at some distance from the chosen loci.

Characterization and evolution of ovine MHC class II DQB sequence polymorphism.

The second exons of OLA-DQB genes from 13 merino sheep were sequenced following amplification by the polymerase chain reaction or isolation from a cDNA library. Ten distinct exon 2 sequences, coding for 10 novel amino acid sequences, were characterized in these sheep. The single-strand conformation polymorphism technique was shown to be capable of discriminating between all sequences. This brings the total number of different OLA-DQB exon 2 sequences (nucleotide and amino acid) which have been characterized to 12, and demonstrates that the OLA-DQB region is highly polymorphic with 29% of nucleotide and 46% of amino acid sites showing variation. Evidence is presented that the OLA-DQB sequences belong to at least two lineages of DQB genes. Some ovine DQB sequences are more like bovine DQB counterparts than other ovine DQB sequences suggesting that the artiodactyl DQB gene and allele lineages predate the separation of the ovine and bovine species 20 million years ago.

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes.

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine lambda gt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class II A sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.

Group-specific component subtypes in the population of Tasmania, Australia.

Plasma specimens from 2,010 blood donors resident throughout the island State of Tasmania, Australia, were examined for group-specific component (GC) sub-types using polyacrylamide gel iso-electric focusing. Subdivision of the island into 8 geographic regions revealed no significant heterogeneity. Comparison of GC allele frequencies with those reported in mainland Australian populations indicated that Tasmanian values lie well within the ranges previously reported.

Serum protein polymorphisms in patients with primary Sjögren's syndrome.

The distribution of 4 serum proteins, the protease inhibitor alpha-1-antitrypsin, haptoglobin, transferrin and group-specific component or vitamin D-binding protein, were examined in 26 patients with primary Sjögren's syndrome and 208 healthy donor controls. No significant associations were found between any of the 4 systems and susceptibility to primary Sjögren's syndrome.

Probing murine male meiosis using unique DNA flanking the immunoglobulin heavy chain genes.

Murine male meiotic preparations were probed by in situ hybridization to localize 10.5 kb of unique DNA which flanks the C gamma 1 heavy chain immunoglobulin gene on the 5' side. The probe, inserted into the plasmid vector pBR 322, was labelled with I125 dCTP using nick translation. After exposing autoradiographs, made by standard techniques of in situ hybridization, for 40 days, a strong signal was obtained on one bivalent of size appropriate to pair 12. The signal was in the distal position on chromosome 12 and it could be traced from spermatogonial divisions through to mature sperm. The signal was strongest in some well-spread stages of prophase of first spermatocytes, weakest in early spermatids, then surprisingly strong in sperm heads. This variation is probably due to differences in the packaging of the target sequence at the various stages of meiosis.

GC subtypes and malignant melanoma.

Using polyacrylamide gel isoelectric focusing (PAGIF), group-specific component (GC) subtypes were determined for 64 malignant melanoma patients and 208 controls residing in Victoria, Australia. No association was found, confirming the results of earlier studies.