Production of functional rat liver PSP protein in Escherichia coli

An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-β-D-galactopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH₂-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver. Received 8 October 1998; received after revision 6 November 1998; accepted 6 November 1998     

Regulation of caldesmon activity by Cdc2 kinase plays an important role in maintaining membrane cortex integrity during cell division

To study the mitosis-specific phosphorylation of caldesmon (CaD), we generated a mutant of the C-terminal fragment (amino acids 244–538) of human fibroblast CaD (CaD39-6F), as well as a mutant of the full-length CaD (CaD-6F), in which all six potential phosphorylation sites for Cdc2 kinase were abolished. The mitotic CaD39-6F-overexpressing cells required more time to progress from anaphase start to 50% cytokinesis, exhibited larger size, and abnormally formed numerous small blebs. In contrast, overexpression of the wild-type C-terminal fragment of CaD (CaD39) did not result in abnormal bleb formation, but led to larger size and prolonged the time requirement between anaphase start and 50% cytokinesis. Similar abnormal blebs were also observed in the CaD-6F-overexpressing cells. CaD-6F-overexpressing cells did not show larger size but required more time to progress from anaphase start to 50% cytokinesis. These results suggest that mitosis-specific phosphorylation of CaD plays a role in inhibiting bleb formation and that the N-terminal fragment of CaD is required for cell size determination. Received 4 September 2002; received after revision 25 November 2002; accepted 4 December 2002     

Short-range linkage relationships, genomic organisation and sequence comparisons of a cluster of five HSP70 genes in Fugu rubripes

Twelve cosmids containing sequences resembling genes encoding members of the 70-kDa heat-shock protein family, HSP70, have been isolated from Fugu rubripes. They can be broadly divided into three groups of overlapping cosmids. Restriction analysis and sequencing of one set of five cosmids have revealed five intronless Fugu HSP70 genes spanning 42 kb, arranged in a combined head-to-head, tail-to-tail and head-to-tail orientation. The levels of DNA and amino acid identity are very high with respect to one another, and are most similar to HSP70 sequences linked to the major histocompatibility complex (MHC) region in other species. Putative heat-shock consensus elements are identified. Non-HSP70 sequences with homology to known genes have been found physically linked to this Fugu HSP70 cluster: the Drosophila melanogaster SOL gene, the Drosophila melanogaster nemo gene, the Caenorhabditis elegans T17E9.1 gene and the sequence encoding the serine protease domain. The linkage relationships described here so far bear no resemblance to those of HSP70 in other organisms. Convergence of mammalian HSP70 and MHC class I and II loci probably occurred after fish had diverged. Received 17 November 1998; received after revision 25 February 1999; accepted 26 February 1999     

Endogenous synthesis of peptidoglycan in eukaryotic cells; a novel concept involving its essential role in cell division,tumor formation and the biological clock

Degradation products of peptidoglycan, the universal bacterial cell wall constituent, were previously found in animal tissues and urine. Reassessment and quantitative analysis of available data lead to an original concept, i.e. that eukaryotic cells synthesize peptidoglycan. We present a model in which this endogenously synthesized peptidoglycan is essential for the processes of eukaryotic cell division and sleep induction in animals. Genes for peptidoglycan metabolism, like those for lysine biosynthesis in plants, are probably inherited from endosymbiotic bacteria, the ancestors of mitochondria and chloroplasts. Corollaries of this concept, i.e. roles for peptidoglycan metabolism in tumor formation and in the biological clock, are supported by abundant evidence. We propose that many interactions between bacteria and eukaryotes are conditioned by their common genetic heritage.     

Ras proteins in the control of the cell cycle and cell differentiation

The Ras family of small GTPases includes three closely related proteins: H-, K-, and N-Ras. Ras proteins are involved in the transduction of signals elicited by activated surface receptors, acting as key components by relaying signals downstream through diverse pathways. Mutant, constitutively activated forms of Ras proteins are frequently found in cancer. While constitutive Ras activation induces oncogenic-like transformation in immortalized fibroblasts, it causes growth arrest in primary vertebrate cells. Induction of p53 and cyclin-dependent kinase inhibitors such as p15^INK4b, p16^INK4a, p19^ARF, and p21^WAF1 accounts for this response. Interestingly, while ras has usually been regarded as a transforming oncogene, the analysis of Ras function in most of the cellular systems studied so far indicates that the promotion of differentiation is the most prominent effect of Ras. While in some cell types, particularly muscle, Ras inhibits differentiation, in others such as neuronal, adipocytic, or myeloid cells, Ras induces differentiation, in some cases accompanied by growth arrest. Several possible mechanisms for the pleiotropic effects of Ras in animal cells are discussed. Received 8 March 2000; received after revision 24 May 2000; accepted 24 May 2000     

The involvement of the renin-angiotensin system in the regulation of cell proliferation in the rat endometrium

Oestrogens are known to enhance angiotensin biosynthesis by increasing the elaboration of its precursor, angiotensinogen. On the other hand, we found that inhibition of angiotensin-converting enzyme (ACE) suppressed the proliferative response of the rat anterior pituitary gland to oestrogens. To answer the question whether the angiotensin system is involved in the control of the cell proliferation of the uterine epithelium, the effects of an ACE inhibitor, enalapril maleate, and of angiotensins II and IV, alone or together with losartan, an antagonist of angiotensin receptor type 1 (AT1), on endometrial epithelial cell proliferation have been studied. The experiments were performed on ovariectomized female Wistar rats. In the first experiment the animals were injected with a single dose of oestradiol benzoate or received an injection of solvent only. Half of the oestrogen-treated rats were injected additionally with enalapril maleate (EN, twice daily). The incorporation of bromodeoxyuridine (BrDU) into endometrial cell nuclei was used as an index of cell proliferation. It was found that oestradiol alone dramatically increased the BrDU labelling index (LI) of endometrial cell nuclei, and this effect was partially blocked by the simultaneous treatment with EN. In the second experiment, the animals were injected intraperitoneally with angiotensin II (AII), angiotensin IV (AIV) or saline, alone or together with losartan. It was found that AIV induced an increase in the LI in uterine epithelium, and this effect was not blocked by the simultaneous treatment with losartan. The increase in LI in uterine epithelium was also observed in the rats treated with AII and with losartan. These findings suggest an involvement of angiotensin IV in the control of uterine epithelium cell proliferation. Received 12 October 1998; received after revision 6 January 1999; accepted 2 February 1999