Mapping of receptor binding sites on IL-1 beta by reconstruction of IL-1ra-like domains.

Upon structure comparison between IL-1 beta and its antagonist IL-1ra, single or multiple residues along the IL-1 beta sequence were replaced with the corresponding amino acids present in the IL-1ra protein, in the attempt to identify sites important for receptor binding and for biologic activity on the two molecules. Ten of fifteen mutant proteins had activity comparable to that of wild-type IL-1 beta in three different biologic assays and in receptor binding, indicating that the introduced changes did not influence the functional structure of the protein. Conversely, three mutants (SMIL-9: 127/263 R/T-->W/Y; SMIL-10: 125/127/263/265 T/R/T/Q-->R/W/Y/E; SMIL-15:222/227 I/E-->S/S) showed an increased binding capacity for IL-1RI, not paralleled by increased agonist activity, indicating that the introduced IL-1ra residues could be involved in the nonagonist IL-1RI binding site. On the other hand, two mutants showed diminished binding capacity with concomitant decrease in biologic activity. Both mutants (SMIL-1, five substitutions in the loop 202-214; and SMIL-3, total replacement of the loop 164-173 with the IL-1ra stretch 52-55) included substitutions of residues allegedly important for agonist binding to IL-1RI. Mutant SMIL-3 showed the most profound reduction in binding capacity for IL-1RI (CDw121a) and a more than 1,000-fold reduced biologic activity both in vitro and in vivo, but it retained full capacity of binding to IL-1RII (CDw121b) and acted as a selective antagonist of IL-1RII. From these results the following conclusions can be drawn. IL-1 beta binds to IL-1RI and to IL-1RII through different sites, and the loop 164-173 appears as one of the areas involved in the selective interaction with IL-1RI. Agonist (IL-1 beta) and nonagonist (IL-1ra) binding to IL-1RI occur through distinct sites, with loops 164-173 and 202-214 of IL-1 beta identified as two of the sites selectively involved in agonist binding to the activating receptor.

The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools.

The CDC25 gene is transcribed at a very low level in S. cerevisiae cells. We have studied the effects of an overexpression of this regulatory gene by cloning either the whole CDC25 open reading frame (pIND25-2 plasmid) or its 3' terminal portion (pIND25-1 plasmid) under the control of the inducible strong GAL promoter. The strain transformed with pIND25-2 produced high levels of CDC25 specific mRNA, induced by galactose. This strain does not show any apparent alteration of growth, both in glucose and in galactose. Instead the yeast cells transformed with pIND25-1, that overexpress the 3' terminal part of CDC25 gene, grow very slowly in galactose medium, while they grow normally in glucose medium. The nucleotides were extracted from transformed cells, separated by HPLC and quantitated. The ATP/ADP and GTP/GDP ratios were almost identical in control and in pIND25-2 transformed strains growing in glucose and in galactose, while the strain that overexpresses the 3' terminal portion of CDC25 gene showed a decrease of ATP/ADP ratio and a partial depletion of the GTP pool. The disruption of RAS genes was only partially able to 'cure' this phenotype. A ras2-ts1, ras1::URA3 strain, transformed with pIND25-1 plasmid, was able to grow in galactose at 36 degrees C. These results suggest that the carboxy-terminal domain of the CDC25 protein could stimulate an highly unregulated GTPase activity in yeast cells by interacting not only with RAS gene products but also with some other yeast G-proteins.

Production processes of recombinant IL-1 beta from Bacillus subtilis: comparison between intracellular and exocellular expression.

The human IL-1 beta coding sequence derived from a cDNA library was inserted into two different plasmid expression vectors, pSM214 and pSM308, which promote the synthesis of recombinant products intracellularly and exocellularly, respectively. The hybrid constructs pSM261 and pSM320 were obtained. Bacillus subtilis SMS118 was transformed with these plasmids and the recombinant strains were grown in 1 litre bioreactors. Different growth conditions were analyzed to optimize yields both in terms of biomass and IL-1 beta production. In the pSM261-harbouring strain, IL-1 beta was synthesized in the cytoplasm to levels ranging from 1 to 2.7 mg g-1 of cells, corresponding to up to 40 mg l-1 of the culture. In contrast, SMS118(pSM320) was able to secrete 0.27 mg of natural IL-1 beta per g of cells (6.7 mg l-1 of culture). Processes for the purification of IL-1 beta from the supernatant and the biomass of the two cultures were also developed and compared in terms of yield and simplicity of the purification schemes. From our data it turns out that the route of intracellular expression is very efficient and superior to the one which results in secretion of IL-1 beta. This indicates that the use of B. subtilis as a recombinant host in biotechnology is not strictly dependent on its ability to secrete proteins into the culture medium.

The glucose-induced polyphosphoinositides turnover in Saccharomyces cerevisiae is not dependent on the CDC25-RAS mediated signal transduction pathway.

Recently the polyphosphoinositides (PI) turnover has been related to the control of growth and cell cycle also in Saccharomyces cerevisiae, and the RAS2 and RAS1 gene products have been shown to be involved in the stimulation of PI turnover in G0/G1 arrested yeast cells. Here we show that addition of glucose to previously glucose-starved cells, stimulates, the PI turnover with fast kinetics also in yeast cells that were not arrested in the G0/G1 phase of the cell cycle. In addition PI turnover is equally stimulated in temperature sensitive cdc25-1 and cdc25-5 strains at restrictive temperature, as well as in ras1, ras2-ts strain, suggesting that PI turnover stimulation is not dependent on the CDC25-RAS mediated signal transduction pathway.

Overexpression of the CDC25 gene, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, allows immunological identification and characterization of its gene product.

The product of the START gene CDC25, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, was identified using specific antibodies raised against a chimeric beta-galactosidase/CDC25 protein. The CDC25 protein is poorly expressed and can be detected only when the CDC25 gene is overexpressed under the control of the galactose-inducible GAL1-10 strong promoter elements. It has a molecular weight of 180,000, is not glycosylated and is strongly associated with the particulate fraction. After deletion of residues 1255-1550 the protein is found in the soluble fraction.

Characterization of transformed cell lines evolving from a normal C3H line.

The development of transformed cell lines evolving from an embryo fibroblastic C3H primary culture was followed before and after the ageing crisis using different techniques. By flow cytometry, alteration of subpopulations having different DNA content and altered metabolic activity was observed after the crisis, with the trend to assume a near tetraploid DNA index at higher passages. The fibrin clot retractile activity was lost in all cases during the ageing crisis, but the outcome did not present uniform values of growth characteristics or chromosome number and tumorigenicity appeared to be a nonstable property of the transformed cell lines.

Effects of temperature on the yeast cell cycle analyzed by flow cytometry.

The effects of temperature (in the range 15-36 degrees C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30 degrees C, and a transition point is apparent at about 20 degrees C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21-36 degrees C, while duration of G2+ M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell-cycle model for budding yeast.