Expression of AML1-d, a short human AML1 isoform, in embryonic stem cells suppresses in vivo tumor growth and differentiation.

The human AML1 gene encodes a heterodimeric transcription factor which plays an important role in mammalian hematopoiesis. Several alternatively spliced AML1 mRNA species were identified, some of which encode short protein products that lack the transactivation domain. When transfected into cells these short isoforms dominantly suppress transactivation mediated by the full length AML1 protein. However, their biological function remains obscure. To investigate the role of these short species in cell proliferation and differentiation we generated embryonic stem (ES) cells overexpressing one of the short isoforms, AML1-d, as well as cells expressing the full length isoforms AML1-b and AML2. The in vitro growth rate and differentiation of the transfected ES cells were unchanged. However, overexpression of AML1-d significantly affected the ES cells' ability to form teratocarcinomas in vivo in syngeneic mice, while a similar overexpression of AML1-b and AML2 had no effect on tumor formation. Histological analysis revealed that the AML1-d derived tumors were poorly differentiated and contained numerous apoptotic cells. These data highlight the pleiotropic effects of AML1 gene products and demonstrate for the first time an in vivo growth regulation function for the short isoform AML1-d.

Restoration of a normal level of immunoglobulin production in a hybridoma cell line following modification of the chromosomal immunoglobulin mu gene by gene replacement.

In the present investigation, we have measured the levels of IgM mRNA and protein in recombinant cell lines in which the chromosomal immunoglobulin mu gene has been modified by gene replacement (gene conversion or double reciprocal recombination) or vector integration (single reciprocal recombination) events. Our studies reveal that chromosomal immunoglobulin mu genes modified by gene replacement are expressed at wild-type levels whereas those modified by vector integration have lower levels of immunoglobulin mu gene expression. These results suggest that gene replacement may a preferred method for the construction of hybridoma and myeloma cell lines producing optimized immunoglobulins and for studies of immunoglobulin gene function.

Retinoic acid stimulates the synthesis of a novel heat shock protein in the regenerating forelimb of the newt.

Morphogenetic effects of retinoic acid (RA) on the urodele amphibian limb regenerate pattern have been well documented, but little is known regarding the mechanism of this action of RA at the molecular level. Since exogenous RA, at concentrations sufficient to cause proximalization, represents a significant stress to newts and has been shown previously to elicit increased synthesis of heat shock proteins (HSPs) in mouse embryo limb buds, we investigated the effects of this putative morphogen on the synthesis of members of the 70-kilodalton (70-kDa) stress protein family in amputated forelimbs of the newt Notophthalmus viridescens. Injection (i.p.) of RA in dimethyl sulfoxide (DMSO), at a dose sufficient to cause significant proximal-distal reduplication of the pattern in 50% of animals treated, resulted in increased synthesis and accumulation of a 73-kDa protein with a pI of approximately 6.75. The synthesis of this same protein is increased in limb tissues as a result of a brief 35 degrees C heat shock. This protein is electrophoretically distinct from the newt HSP 70 family members, displays a different partial peptide map, and shows no immunological cross-reactivity with an anti-human HSP 70 monoclonal antibody. It may be a member of a separate family of 70- to 73-kDa HSPs. Interestingly, the synthesis of this protein is increased and it is more abundant in control, proximal moderate-early bud stage regenerates at 6 days after i.p. injection of DMSO than in similarly treated distal regenerates. This protein is, in addition, increased in distal regenerates to proximal levels by a prior injection of RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of the DNA sequence-specific binding properties of heat shock transcription factor in Xenopus laevis embryos.

The binding of heat shock transcription factor (HSF) to the heat shock element (HSE) is necessary for transcriptional activation of eukaryotic heat shock protein (HSP) genes. The properties of Xenopus embryo HSF were examined by DNA mobility shift analysis employing a synthetic oligonucleotide corresponding to the proximal HSE in the promoter of the Xenopus HSP70B gene. Heat shock induced activation of HSF binding in Xenopus neurulae was not affected by an inhibition of protein synthesis, indicating that the mode of activation may be posttranslational. Also, while HSF binding was activated in control Drosophila cell extracts by in vitro heat shock or other chemical treatments, HSF binding in Xenopus embryo or somatic cell extract was not. Thus, the activation of Xenopus HSE-HSF binding may occur via a different mechanism compared with Drosophila. Furthermore, we determined that the native size of heat-induced HSF in pre- and post-midblastula stage Xenopus embryos is approximately 530 kilodaltons (kDa), which corresponds to a hexamer made up of 88 kDa monomers. Finally, the slower accumulation of HSP70 mRNA to peak levels found at lower heat shock temperatures was not correlated with HSE-HSF binding activity.

Analysis of CCAAT box transcription factor binding activity during early Xenopus laevis embryogenesis.

Unfertilized eggs and pre-midblastula (MBT) stage Xenopus embryos were found to contain a large pool of maternally derived CCAAT box-binding transcription factor (CBTF). DNA mobility shift experiments using embryonic extracts prepared with either low or high salt buffers suggest that Xenopus CBTF may not interact with embryonic DNA until the late blastula stage, a time point coincident with the increase in zygotic transcription. Additionally, photoaffinity-labeling experiments revealed that both pre- and post-MBT CBTF-binding activities were composed of at least three proteins having relative molecular masses of 68, 52, and 42 kDa.

Antibiotic sensitivity of pathogens from infected skin lesions.

One hundred and seven patients attending an accident and emergency department with infected skin lesions were studied. The commonest organisms isolated were Staph aureus and Strep pyrogenes. Routine antimicrobial sensitivity testing of bacterial isolates showed that the antibiotic most likely to be effective against the organisms isolated was a cephalosporin or a tetracycline.