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The receptors for insulin and insulin-like growth factor-I (IGF-I) belong to the family of receptor protein tyrosine kinases [1]. Although a vast body of data supports the concept that insulin stimulates cell growth in vitro and in vivo, the question of whether insulin is physiologically a growth factor remains controversial (for review see [2]). Even more controversial is the question of whether insulin is capable of inducing mitogenic effects through its own receptor, or whether the growth-promoting effects of insulin result from its weak interaction with the IGF-I receptor or occur within insulin/IGF-I receptor hybrids [3,4], or via interphosphorylation of the IGF-I receptor by the insulin receptor tyrosine kinase [5]. The response possibly depends on the cell type and its given supply of insulin and IGF-I receptors as well as the subsets of intracellular signalling molecules that are activated by either receptor. (We use the term IGF-I receptor for simplicity to designate the type 1 IGF receptor which binds both IGF-I and II and probably mediates the mitogenic effects of both growth factors [6].) Diabetologia (1997) Summary Insulin has traditionally been considered as a hormone essential for metabolic regulation, while the insulin-like growth factors (IGF-I and IGF-II) are postulated to be more specifically involved in growth regulation. The conventional wisdom is that they share each other's effects only at high concentrations, due to their weak affinity for the heterologous receptor. We discuss here the evidence that in the proper cellular context, insulin can be mitogenic at physiologic concentrations through its own receptor. We studied the insulin and IGF-I binding characteristics of a new model suitable for analysing insulin receptor mediated mitogenesis; that is, a T-cell lymphoma line that depends on insulin for growth, but is unresponsive to IGFs. The cells showed no specific binding of 125 I-IGF-I and furthermore, no IGF-I receptor mRNA was detected by RNAse protection assay in the LB cells, in contrast with mouse brain and thymus. The cells bound at saturation about 3000 insulin molecules to receptors that had normal characteristics in terms of affinity, kinetics, pH dependence and negative co-operativity. A series of insulin analogues competed for 125 I-insulin binding with relative potencies comparable to those observed in other insulin target cells. The full sequence of the insulin receptor cDNA was determined and found to be identical to the published sequence of the murine insulin receptor cDNA. The LB cell line is therefore an ideal model with which to investigate insulin mitogenic signalling without interference from the IGF-I receptor. Using this model, we have started approaching the molecular basis of insulin-induced mitogenesis, in particular the role of signalling kinetics in choosing between mitogenic and metabolic pathways. [Diabetologia (1997) 40: S 25-S 31]

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlumydomonus reinhurdtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine a-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.In response to a sudden elevation of temperature, a number of specific genes, termed heat-shock genes, are activated in both procaryotic and eucaryotic cells [ 11. Following induction, a rapid transcription and preferential translation of the corresponding heat-shock mRNAs takes place and results in a significant accumulation of the heat-shock proteins [2]. In accordance with the universality of the heat-shock response, a relatively high degree of similarity characterizes heat-shock proteins from various organisms. In higher plants smallmolecular-mass heat-shock proteins (1 5 -30 kDa) represent a particularly prominent and heterogeneous group of proteins coded by multigene families [3 -61. The complexity and the diversity in the sizes of the low-molecular-mass heat-shock proteins seem to indicate a lower degree of similarity, in contrast to the high degree of conservation in the amino acid sequences of the high-molecular-mass heat-shock proteins common to both the plant and animal kingdoms. Like their high-molecular-mass counterparts, the small heat-shock proteins in plants are nuclear-coded and translated on cytosolic ribosomes. The majority of them remain in the cytosol and become associated with the cytoskeleton...

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Expression of the nuclear coded heat shock protein HSP22 in Chlamydomonas reinhardtii y‐1 cells is light regulated at the level of transcript accumulation. In dark grown cells, containing a non‐differentiated plastid, light has an additional regulatory effect on the accumulation of HSP22. When such cells are exposed to heat stress in the light, poly(A)+ RNA hybridizing with the HS22 probe is synthesized at levels comparable with those found in cells pre‐illuminated for 3 h (greening) prior to the heat shock. However, this RNA is poorly translated in vitro and HSP22 does not accumulate in vivo. HS22 mRNA efficiently translated in vitro is induced in dark grown cells only when chloroplast differentiation has been initiated by exposure to the light for 3 h. In these cells HSP22 accumulates during heat shock. Inhibition of plastid translation activity during light‐dependent chloroplast development prevents accumulation of HSP22 in vivo. However the HS22 mRNA formed in this case can be efficiently translated in vitro. Light requirement for the accumulation of HSP22 during heat stress is exhibited also by wild type C. reinhardtii cells which possess a differentiated chloroplast irrespective of the light conditions during cell growth. However dark grown wild type cells do not require pre‐illumination for developing the ability to accumulate HSP22 during heat stress in the light.

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.

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