Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.

Cerebral organization for language in deaf and hearing subjects: Biological constraints and effects of experience

Cerebral organization during sentence processing in English and in American Sign Language (ASL) was characterized by employing functional magnetic resonance imaging (fMRI) at 4 T. Effects of deafness, age of language acquisition, and bilingualism were assessed by comparing results from (i) normally hearing, monolingual, native speakers of English, (ii) congenitally, genetically deaf, native signers of ASL who learned English late and through the visual modality, and (iii) normally hearing bilinguals who were native signers of ASL and speakers of English. All groups, hearing and deaf, processing their native language, English or ASL, displayed strong and repeated activation within classical language areas of the left hemisphere. Deaf subjects reading English did not display activation in these regions. These results suggest that the early acquisition of a natural language is important in the expression of the strong bias for these areas to mediate language, independently of the form of the language. In addition, native signers, hearing and deaf, displayed extensive activation of homologous areas within the right hemisphere, indicating that the specific processing requirements of the language also in part determine the organization of the language systems of the brain.

Reverse biochemistry: Use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a “fold-specific” inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription–PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Cation-pi interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide.

The cation-pi interaction is an important, general force for molecular recognition in biological receptors. Through the sidechains of aromatic amino acids, novel binding sites for cationic ligands such as acetylcholine can be constructed. We report here a number of calculations on prototypical cation-pi systems, emphasizing structures of relevance to biological receptors and prototypical heterocycles of the type often of importance in medicinal chemistry. Trends in the data can be rationalized using a relatively simple model that emphasizes the electrostatic component of the cation-pi interaction. In particular, plots of the electrostatic potential surfaces of the relevant aromatics provide useful guidelines for predicting cation-pi interactions in new systems.

An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness.

While studies of the regulation of gene expression have generally concerned qualitative changes in the selection or the level of expression of a gene, much of the regulation that occurs within a cell involves the continuous subtle optimization of the levels of proteins used in macromolecular complexes. An example is the biosynthesis of the ribosome, in which equimolar amounts of nearly 80 ribosomal proteins must be supplied by the cytoplasm to the nucleolus. We have found that the transcript of one of the ribosomal protein genes of Saccharomyces cerevisiae, RPL32, participates in such fine tuning. Sequences from exon I of the RPL32 transcript interact with nucleotides from the intron to form a structure that binds L32 to regulate splicing. In the spliced transcript, the same sequences interact with nucleotides from exon II to form a structure that binds L32 to regulate translation, thus providing two levels of autoregulation. We now show, by using a sensitive cocultivation assay, that these RNA structures and their interaction with L32 play a role in the fitness of the cell. The change of a single nucleotide within the 5' leader of the RPL32 transcript, which abolishes the site for L32 binding, leads to detectably slower growth and to eventual loss of the mutant strain from the culture. Experiments designed to assess independently the regulation of splicing and the regulation of translation are presented. These observations demonstrate that, in evolutionary terms, subtle regulatory compensations can be critical. The change in structure of an RNA, due to alteration of just one noncoding nucleotide, can spell the difference between biological success and failure.

Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene

With the aim of improving the nutritive value of an important grain legume crop, a chimeric gene specifying seed-specific expression of a sulfur-rich, sunflower seed albumin was stably transformed into narrow-leafed lupin (Lupinus angustifolius L.). Sunflower seed albumin accounted for 5% of extractable seed protein in a line containing a single tandem insertion of the transferred DNA. The transgenic seeds contained less sulfate and more total amino acid sulfur than the nontransgenic parent line. This was associated with a 94% increase in methionine content and a 12% reduction in cysteine content. There was no statistically significant change in other amino acids or in total nitrogen or total sulfur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization, compared with wild-type seeds. These findings demonstrate the feasibility of using genetic engineering to improve the nutritive value of grain crops.

Empirical laws of survival and evolution: Their universality and implications

Presented analysis of human and fly life tables proves that with the specified accuracy their entire survival and mortality curves are uniquely determined by a single point (e.g., by the birth mortality q0), according to the law, which is universal for species as remote as humans and flies. Mortality at any age decreases with the birth mortality q0. According to life tables, in the narrow vicinity of a certain q0 value (which is the same for all animals of a given species, independent of their living conditions), the curves change very rapidly and nearly simultaneously for an entire population of different ages. The change is the largest in old age. Because probability to survive to the mean reproductive age quantifies biological fitness and evolution, its universal rapid change with q0 (which changes with living conditions) manifests a new kind of an evolutionary spurt of an entire population. Agreement between theoretical and life table data is explicitly seen in the figures. Analysis of the data on basic metabolism reduces it to the maximal mean lifespan (for animals from invertebrates to mammals), or to the maximal mean fission time (for bacteria), and universally scales them with the total number of body atoms only. Phenomenological origin of this unification and universality of metabolism, survival, and evolution is suggested. Their implications and challenges are discussed.