Novel CT domain-encoding splice forms of CTGF/CCN2 are expressed in B-lineage acute lymphoblastic leukaemia.

INTRODUCTION: Connective tissue growth factor (CTGF/CCN2) has been shown previously to be aberrantly expressed in a high proportion of paediatric precursor B cell acute lymphoblastic leukaemia (pre-B ALL), suggesting a potential oncogenic role in this tumour type. We therefore assessed CTGF mRNA transcript diversity in B-lineage ALL using primary patient specimens and cell lines. METHODS: CTGF mRNA expression was evaluated by quantitative real-time PCR and Northern blotting. We performed a structural analysis of CTGF mRNA by nested reverse-transcriptase PCR and examined CTGF protein diversity by immunoblotting. RESULTS: Northern blot analysis of pre-B ALL cell lines revealed short CTGF transcripts that were expressed in association with the active phase of cellular growth. Structural analysis confirmed the synthesis of several novel CTGF mRNA isoforms in B-lineage ALL cell lines that were uniformly characterised by the retention of the coding sequence for the C-terminal (CT) domain. One of these novel spliceforms was expressed in a majority (70%) of primary pre-B ALL patient specimens positive for canonical CTGF mRNA. Evidence that these alternative transcripts have coding potential was provided by cryptic CTGF proteins of predicted size detected by immunoblotting. CONCLUSION: This study identifies for the first time alternative splicing of the CTGF gene and shows that a short CTGF splice variant associated with cell proliferation is expressed in most cases of primary CTGF-positive pre-B ALL. This novel variant encoding only the CT domain may play a role in pre-B ALL tumorigenesis and/or progression.

Survival of motor neuron protein over-expression prevents calpain-mediated cleavage and activation of procaspase-3 in differentiated human SH-SY5Y cells.

Spinal muscular atrophy (SMA), a neurodegenerative disorder primarily affecting motor neurons, is the most common genetic cause of infant death. This incurable disease is caused by the absence of a functional SMN1 gene and a reduction in full length survival of motor neuron (SMN) protein. In this study, a neuroprotective function of SMN was investigated in differentiated human SH-SY5Y cells using an adenoviral vector to over-express SMN protein. The pro-survival capacity of SMN was assessed in an Akt/PI3-kinase inhibition (LY294002) model, as well as an oxidative stress (hydrogen peroxide) and excitotoxic (glutamate) model. SMN over-expression in SH-SY5Y cells protected against Akt/phosphatidylinositol 3-kinase (PI3-kinase) inhibition, but not oxidative stress, nor against excitotoxicity in rat cortical neurons. Western analysis of cell homogenates from SH-SY5Y cultures over-expressing SMN harvested pre- and post-Akt/PI3-kinase inhibition indicated that SMN protein inhibited caspase-3 activation via blockade of calpain-mediated procaspase-3 cleavage. This study has revealed a novel anti-apoptotic function for the SMN protein in differentiated SH-SY5Y cells. Finally, the cell death model described herein will allow the assessment of future therapeutic agents or strategies aimed at increasing SMN protein levels.

Association of TGIFLX/Y mRNA expression with prostate cancer.

Prostate cancer is the most common type of solid tumor and a leading cause of cancer-related death of men living in the developed world. In recent years, the molecular mechanisms involved in prostate cancer development and/or progression have been intensely studied and several genes have been identified. TGIFLX/Y (TGIFLX and TGIFLY) are members of the homeobox superfamily of genes whose function(s) is unknown. To investigate TGIFLX/Y mRNA expression in prostate cancer, we studied two different types of clinical samples, namely 60 prostate tumors and 15 cases of benign prostate hyperplasia (BPH), by RT-PCR. Our results revealed that most prostate tumors (73.5%) express at least one of these genes, although different patterns of TGIFLX/Y mRNA expression were observed. In some tumor samples the expression of both genes was detected, while in others no expression of either gene was observed. Notably, there was a significant correlation between expression of both TGIFLX and TGIFLY and a Gleason score of >or=6 (P = 0.038). By contrast, expression of TGIFLX/Y mRNA in BPH samples could not be detected. These results suggest an association of TGIFLX/Y expression with the progression of prostate cancer.

The nuclear oncoprotein TLX1/HOX11 associates with pericentromeric satellite 2 DNA in leukemic T-cells.

TLX1/HOX11, a DNA-binding homeodomain protein, was originally identified by virtue of its aberrant expression in T-cell leukemia and subsequently found to be crucial for normal spleen development. The precise mechanism of TLX1 function remains poorly understood, although it is known that it can act as both a transcriptional activator and repressor and can downregulate the Aldh1a1 gene in embryonic mouse spleen. Using a whole-genome PCR approach, we show here that TLX1 protein directly interacts with pericentromeric human satellite 2 DNA sequences. Such DNA is known to localize to heterochromatin, which among other roles has been implicated in gene silencing. The interaction was confirmed in vitro and in vivo by gel retardation and chromatin immunoprecipitation assays involving satellite 2 DNA, which contained sequences resembling TLX1 binding sites. Using immunofluorescence microscopy, TLX1 demonstrated a punctate pattern of staining in the nuclei of leukemic T-cells (ALL-SIL). Double labelling indicated that TLX1 colocalized with the centromeric protein CENP-B, demonstrating that the TLX1 foci corresponded to clusters of centromeric DNA. The novel interaction of TLX1 with constitutive heterochromatin adds an additional level of complexity to the intracellular functions of this transcriptional regulator and may have relevance to its roles in transcriptional repression and T-cell immortalization.

Genomic structure, tissue expression and chromosomal location of the LIM-only gene, SLIM1.

Human SLIM1 is a recently described gene of the LIM-only class encoding four and a half tandemly repeated LIM domains. LIM domains are double zinc finger structures which provide an interface for protein/protein interactions and are conserved in a variety of nuclear and cytoplasmic factors important in cell fate determination and cellular regulation. Here we report the structural organization, expression pattern and chromosomal localization of the human SLIM1 gene. SLIM1 was found to contain at least five exons with all four introns disrupting the coding region at a similar position relative to the respective complete LIM domains. Northern blot analysis confirmed strikingly high expression of SLIM1 in skeletal muscle and heart, with much lower expression observed in several other tissues including colon, small intestine and prostate. The SLIM1 gene was assigned to human chromosome Xq26 using fluorescence in situ hybridization.

The T-cell oncogenic protein HOX11 activates Aldh1 expression in NIH 3T3 cells but represses its expression in mouse spleen development.

Hox11 is a homeobox gene essential for spleen formation in mice, since atrophy of the anlage of a developing spleen occurs in early embryonic development in Hox11 null mice. HOX11 is also expressed in a subset of T-cell acute leukemias after specific chromosomal translocations. Since the protein has a homeodomain and can activate transcription, it probably exerts at least some of its effects in vivo by regulation of target genes. Representational difference analysis has been used to isolate cDNA clones corresponding to mRNA species activated following stable expression of HOX11 in NIH 3T3 cells. The gene encoding the retinoic acid-synthesizing enzyme aldehyde dehydrogenase 1 (Aldh1), initially called Hdg-1, was found to be ectopically activated by HOX11 in this system. Study of Aldh1 gene expression during spleen development showed that the presence of Aldh1 mRNA inversely correlated with Hox11. Hox11 null mouse embryos have elevated Aldh1 mRNA in spleen primordia prior to atrophy, while Aldh1 seems to be repressed by Hox11 during organogenesis of the spleens of wild-type mice. This result suggests that expression of Aldh1 protein is negatively regulated by Hox11 and that abnormal expression of Aldh1 in Hox11 null mice may cause loss of splenic precursor cells by aberrant retinoic acid metabolism.

Optimal activation of an endogenous gene by HOX11 requires the NH2-terminal 50 amino acids.

The HOX11 homeobox gene was first identified through studies of the t(7;10) and t(10;14) chromosomal translocations of acute T-cell leukemia. In addition, analysis of Hox11-/- mice has demonstrated a critical role for this gene in murine spleen development. A possible mode of in vivo function for the HOX11 protein in these two situations is regulation of target genes following DNA binding via the homeodomain, but little is known about how HOX11 regulates transcription in vivo. By performing transcriptional studies in yeast and mammalian one-hybrid systems, a modular transcriptional transactivation region at the NH2 terminus of HOX11 has been functionally dissected from other parts of the protein. This NH2-terminal region includes the previously identified short conserved Hep motif, which itself activates transcription in one-hybrid assays. The importance of the NH2-terminal region for the function of HOX11 in vivo was assayed by activating a HOX11-dependent gene in NIH 3T3 cells. Activation of this gene was found to be dependent upon an intact homeodomain in HOX11, but maximal activation was obtained only when the NH2-terminal 50 amino acids of HOX11 was present, showing that this region of HOX11 is important for in vivo transcriptional control of a chromosomal target gene.

Effect on humoral tolerance (IgG and IgE) in dogs by oral administration of ovalbumin and Der pI.

The effect of oral administration of ovalbumin (OVA) or recombinant house dust mite allergen (Der p I) to dogs upon specific IgG and IgE reactions to subcutaneous immunization with these antigens was studied. Daily feeding of 10 x 10 g of OVA resulted in a non-responsiveness to subsequent parenteral immunization with OVA in two young dogs. The same two dogs were also immunized parenterally with Der p I and showed a pronounced IgG response against native Der p I, confirming that the non-responsiveness to OVA was antigen-specific. Thus, it has been demonstrated that it is possible to induce oral tolerance in dogs. Two other dogs of the same litter that received 2 x 10 mg of recombinant Der p I in a crude yeast lysate per os reacted to immunization with OVA with pronounced IgG and IgE production against OVA, further confirming the antigen-specificity of the OVA tolerance. However, tolerance to Der p I was not induced, as evidenced by a strong IgG response to immunization after per os application of the antigen, possibly because the oral dose was too small.

Prevalence of hepatitis C virus sequence variants in South-East Asia.

The nature and distribution of hepatitis C virus (HCV) genotypic variants present in south-east Asia have not been extensively investigated. We analysed HCV RNA obtained from 67 clinical serum samples from Singapore, Thailand, Indonesia, the Philippines and South Korea. All samples were amplified by semi-nested RT-PCR and the nucleotide sequence determined for four regions within the E1, E2/NS1, NS4 and NS5 genes. Each isolate had a unique nucleotide and deduced amino acid sequence, consistent with the genetic heterogeneity of this virus. There was remarkably little amino acid sequence variation between isolates of the same genotype, apart from variable domains within putative envelope glycoproteins that are likely to be under immune pressure. All isolates could be classified according to the currently recognized genotypes of HCV, with the exception of one Singapore isolate that defined a new group 3 subtype. The 1b genotype, which predominates in Japan, was the most widely distributed genotype and accounted for 58% of all isolates sequenced. Regional variations in HCV genotype distribution were observed, with type 3a being found almost exclusively in Thailand. By contrast, the 1a genotype, which predominates in the USA was the most prevalent genotype in the Philippines. Genotype 1a was found less commonly among the Thai isolates, presumably having been introduced from the West in stored blood products or by sporadic transmission. The significant prevalence of HCV types 2 and 3 restates the need for variant genotypes to be included in immunodiagnostic and vaccine development strategies. This study reveals that the 1b genotype of HCV, previously found to be the major variant present in east Asia, also predominantes in the south-east Asian region, and may be the major HCV type found worldwide.

The induction of in vivo superinfection and recombination using feline immunodeficiency virus as the model.

This study investigated the hypothesis that under certain conditions, superinfection of cats with feline immunodeficiency virus (FIV), may occur. One FIV isolate (T91) was used to inoculate three FIV and FeLV-free cats. Blood from an FIV-infected cat (N), which contained two variants and differed from T91 by at least 5% in nucleotide sequence in the env gene, was inoculated into a fourth cat. Both T91 and blood from N were inoculated simultaneously into a fifth cat. After 22 weeks, two of the three cats initially infected with T91 were challenged with blood from N. At 30 weeks following initial infection, peripheral blood mononuclear cells were obtained from all cats, DNA was extracted, and a segment of the env gene was PCR amplified, cloned and sequenced. Nucleotide sequence analysis of the cloned PCR product showed that virus strains used in initial infection were recovered from cats not challenged with a second variant. Challenge of cats with the blood of N following initial infection with T91 resulted in superinfection occurring in one cat and recombination occurring in the other. Furthermore, the use of blood as a source of challenge, in cats where superinfection and simultaneous infections were attempted, may have induced the appearance of variants which more closely resembled the most heterologous strain present in the infectious source.

RAPD (random amplified polymorphic DNA) analysis of Giardia DNA and correlation with isoenzyme data.

Fourteen Giardia duodenalis isolates were examined using the RAPD (random amplified polymorphic deoxyribonucleic acid) technique. Simple reproducible polymorphisms were generated using 3 different RAPD primers. The results generated by each primer were very similar and were significantly correlated with each other. These data were then compared to existing isoenzyme electrophoresis data on the same isolates. The RAPD data divided the isolates into 10 groupings or rapdemes while the isoenzyme data divided them into 10 similar zymodemes. Both methods grouped 4 isolates (BAH42, BAH44c9, BAH12c9 and BAH39c7), which comprised a phenotypically heterogeneous assemblage with respect to growth rate and metabolism, into similar groupings. The 2 methods were significantly correlated (P < 0.001). It will therefore be possible to use RAPD for the characterization of isolates of Giardia, and other parasites such as Cryptosporidium, which are refractory to cultivation in vitro.

Isolation and in vitro translation of messenger RNA encoding allergens of the cat flea, Ctenocephalides felis.

Polyadenylated [poly(A)+] mRNA was isolated from the cat flea, Ctenocephalides felis by oligothymidylic acid-cellulose spin column chromatography and translated in vitro using a cell-free rabbit reticulocyte system. The relative incorporation of 35S-methionine into trichloroacetic acid-precipitable translation products obtained using poly (A+) mRNA was 48.5-fold over control translations performed without added mRNA. SDS-PAGE analysis of the translation products in combination with autoradiography showed that many proteins with apparent molecular weights in the range 14-90 K were synthesized. Immunoprecipitation studies performed using flea-allergic dog sera showed that several of the synthesized proteins corresponded with flea allergens. The allergenicity of the lysates was also confirmed by skin testing.

Characterization of allergens of the cat flea, Ctenocephalides felis: detection and frequency of IgE antibodies in canine sera.

Flea allergens, fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose, were identified using 20 flea-allergic dog sera in an enhanced chemiluminescent assay for canine IgE antibodies. At least 15 different flea components in the molecular weight range of 14-150 K bound IgE and every serum demonstrated a different pattern of binding. Three of the components with apparent molecular weights of 25, 40 and 58 K were each bound by at least 40% of the sera. No reactivity was seen when normal dog sera were used. These results demonstrate a greater number of flea allergens and a far greater diversity of the IgE antibody response to flea allergens than has previously been described, and suggest that immediate hypersensitivity may be an important mechanism in the pathogenesis of canine flea allergy.

Feline immunodeficiency virus infection: plasma, but not peripheral blood mononuclear cell virus titer is influenced by zidovudine and cyclosporine.

The plasma and peripheral blood mononuclear cell (PBMC) titer of feline immunodeficiency virus (FIV) in experimentally infected cats was assessed following administration of either zidovudine or cyclosporine. Treatments were begun 24 h post infection (p.i.) and continued for 4 weeks. Zidovudine treatment did not prevent establishment of infection with FIV, but plasma virus titers were significantly lower than controls at 2 weeks p.i. This reduction of plasma virus titer by zidovudine was not maintained at subsequent sampling times. Similarly, cyclosporine treatment initially lowered plasma virus titers at 2 weeks p.i., but at 4 weeks p.i. the plasma virus titers in cyclosporine-treated cats were significantly higher than in the untreated group. In the untreated group, plasma virus titers declined rapidly to an undetectable level by 14 weeks p.i. Neither zidovudine or cyclosporine treatment significantly influenced the titer of FIV in PBMCs. In all groups (untreated, zidovudine and cyclosporine) the titers in PBMC were high for the duration of the experiment. The decline in plasma virus titers in immunocompetent cats combined with the effect of cyclosporine on plasma titers strongly suggest that the immune system plays a major role in clearing FIV from plasma. In contrast, it appears that the immune response has little impact on PBMC virus titers. This shows that for complete assessment of antiviral agents, both cell-free and cell-associated virus titers must be examined. We also suggest that the limitation of viral titers in PBMC may be of critical importance in the control of lentiviral infection.

Nucleotide sequences of Australian isolates of the feline immunodeficiency virus: comparison with other feline lentiviruses.

Proviral DNA from four Australian isolates of feline immunodeficiency virus (FIV) was amplified by PCR and the nucleotide sequence determined for two conserved regions within gag (p15/p24) and pol (RT) genes. Comparison with the nucleotide and deduced amino acid sequence of two previously described U.S. isolates from California (Petaluma and PPR), and a third from Maryland (MD) as well as the Japanese isolate TM2, revealed a close similarity between the Australian and Californian isolates with 95-97% nucleotide and 96-99% amino acid homologies. By contrast, the Maryland and Japanese isolates were more distantly related with only 84-87% nucleotide and 90-94% amino acid homology with either the Australian or Californian isolates. The relationship of the Australian FIV isolates to other domestic isolates as well as eight lentiviral isolates from wild felidae (panthers) published previously, was investigated further by constructing a phylogenetic tree based on the pol sequence. This revealed two subgroups of FIV, an Australian/Californian group and a less tightly clustered Maryland/Japanese group. These results suggest that the genomic variability of FIV is reflected by more than simply geographic distance. Furthermore, the relative genetic homogeneity found between Australian isolates suggest a shorter period of evolution of the virus in Australia than in North America.

Extensive sequence variation of feline immunodeficiency virus env genes in isolates from naturally infected cats.

In an investigation of the evolution of feline immunodeficiency virus (FIV) in vivo, sequential isolates from a persistently infected cat were examined by direct sequencing following amplification of selected subgenomic regions by polymerase chain reaction (PCR). Three isolates, T90, T91, and T92, obtained over a three-year period revealed no changes to regions known to be conserved within gag and pol genes. Additionally, no change occurred within gag and pol in an isolate recovered from a second cat which was experimentally infected with T90. Changes were detected within an N-terminal region of the envelope glycoprotein gp 120 (env). These consisted of point mutations, some of which would result in amino acid substitutions and the predicted amino acid changes tended to cluster within variable domains. Inoculation of T90 into a second cat resulted in a different pattern of mutations than that observed for the three isolates from the first cat. In all cases, virus isolates derived from the same cat were much more highly related to each other (extent of env variation was 0.5-1.5%) than to isolates from other cats (10-12% env variation). The rate of change of FIV was estimated to be 3.4 x 10(-3) nucleotide substitutions per site per year for the env gene and less than 10(-4) nucleotide substitutions per site per year for the gag and pol genes, values concordant with that found for human immunodeficiency virus 1. Both nucleotide and amino acid changes in the gp 120 region were found to be directional, suggesting that selective pressures influence FIV envelope gene sequences.

IgE binding structures of the major house dust mite allergen Der p I.

The group I allergens Der p I and Der f I are potent allergens of mites from the genus Dermatophagoides. IgE radioimmune dot blots and immunoabsorption with recombinant peptides have been used to define areas of antigenicity. Four linear binding regions comprising residues 15-33, 60-80, 81-94 and 101-111 were found in the N terminal domain and one, 155-187, in the C-terminal domain, but direct evidence for their discontinuous nature is shown. Firstly, the binding activity of residues 60-80 required either C- or N-terminal flanking sequences to express reactivity and secondly a discontinuous determinant was directly demonstrated by the two non-overlapping peptides 53-99 and 101-154 which significantly cross absorbed specificities to one another. This also indicates considerable homogeneity in the antibodies recognising these peptides. The IgE binding peptides could be located to equivalent residues on the X-ray crystallographic structure of the homologous proteins actinidin and papain. The residues 81-94 and 101-111 which gave strong reactivity were located on a flexible loop connecting the domains and represent areas in which synthetic peptides could be expected to retain activity.

IgE and IgG binding of peptides expressed from fragments of cDNA encoding the major house dust mite allergen Der p I.

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.

IgE binding studies with large peptides expressed from Der p II cDNA constructs.

The major mite allergen Der p II shows marked resistance to denaturation and is expressed from cDNA in bacteria with almost all of its IgE binding activity. Despite these properties, the IgE binding activity appears to be dependent on maintaining the complete primary structure. Random fragment libraries of cDNA, able to code for up to 93 of the 129 amino acid residue protein, did not express IgE binding peptides. Large overlapping peptides 1-69, 69-129 and 42-117 expressed as the fusions from the glutathione transferase of pGEX vectors only had binding activity with IgE in 15 out of 57 sera, and this was typically weak. Sera from children with atopic dermatitis bound IgE in seven out of eight cases but this was also weak compared with their strong reactivity to intact recombinant Der p II. The inability of such large peptides to form IgE binding structures suggests that the antigenic determinants of Der p II are highly conformational and restricted.

Antigenic analysis of group I house dust mite allergens using random fragments of Der p I expressed by recombinant DNA libraries.

Antigenic regions of a major house dust mite allergen, Der p I, were identified by a recombinant DNA strategy employing the technique of random fragmentation. Fragments of cDNA coding for Der p I were produced by sonication and used to construct lambda gt 11 expression libraries. Analyses of recombinant fragments reactive with a rabbit anti-Der p I antiserum showed that the B cell determinants expressed in Escherichia coli were limited, with the majority (86%) of antigenic clones isolated mapping to the region comprising amino acid sequence position 60-80. To define antigenic regions of Der p I more precisely, selected overlapping fragments were subcloned into the expression vector pGEX-1. Dot blot immunoassay and immunoabsorption studies using individual fusion proteins revealed five regions - 34-47, 60-72, 82-99, 112-140, and 166-194 - to contain B cell determinants responsible for the antigenicity of recombinant Der p I. Absorption of the antiserum with Dermatophagoides pteronyssinus extract removed reactivity to all fragments, whereas absorption with an extract from the related mite Dermatophagoides farinae removed reactivity to peptides containing residues 34-47, 60-72, and 166-194, but not 82-99 and 112-140. Similarly, rabbit anti-D.farinae reacted strongly with peptides containing residues 34-47, 60-72, and 166-194, but not residues 82-99 and 112-140 which again showed antigenic differences in these residues between the group I allergens.