Primary structure of a non-secretory ribonuclease from bovine kidney

The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).     

Primary structure of a ribonuclease from bovine brain

The primary structure of a pyrimidine base-specific ribonuclease from bovine brain was determined. The sequence determined is (sequence; see text). Although the sequence homology of this RNase with bovine pancreatic RNase A is 78.2%, it consists of 140 amino acid residues, and it is 16 amino acid residues longer than RNase A at the carboxyl-terminal. In addition to an N-glycosylated long carbohydrate chain, the bovine brain RNase has two short O-glycosylated carbohydrate chains at the 129th and the 133rd serine residues. The additional C-terminal tail of the bovine brain RNase has a unique composition: 6 proline, 5 hydrophobic amino acids, and two basic amino acids, arginine and histidine.     

Memory CD8+ T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

The antigens recognized by individual CD8(+) T cells are small peptides bound to major histocompatibility complex (MHC) class I molecules. The CD8(+) T cell response to a virus is restricted to several peptides, and the magnitudes of the effector as well as memory phases of the response to the individual peptides are generally hierarchical. The peptide eliciting a stronger response is called immunodominant (ID), and those with smaller-magnitude responses are termed subdominant (SD). The relative importance of ID and SD determinants in protective immunity remains to be fully elucidated. We previously showed that multispecific memory CD8(+) T cells can protect susceptible mice from mousepox, an acute lethal viral disease. It remained unknown, however, whether CD8(+) T cells specific for single ID or SD peptides could be protective. Here, we demonstrate that immunization with dendritic cells pulsed with ID and some but not all SD peptides induces memory CD8(+) T cells that are fully capable of protecting susceptible mice from mousepox. Additionally, while natural killer (NK) cells are essential for the natural resistance of nonimmune C57BL/6 (B6) to mousepox, we show that memory CD8(+) T cells of single specificity also protect B6 mice depleted of NK cells. This suggests it is feasible to produce effective antiviral CD8(+) T cell vaccines using single CD8(+) T cell determinants and that NK cells are no longer essential when memory CD8(+) T cells are present.     

An insight into the genetic polymorphism among European populations of Lactuca serriola assessed by AFLP

Prickly lettuce (Lactuca serriola) is world-wide distributed and very variable species generally considered as a progenitor of the cultivated lettuce (Lactuca sativa). Altogether, 50 populations of L. serriola were characterized by means of amplified fragment length polymorphism (AFLP) and by isozyme analysis. Relationships among individuals and populations were examined by applying the unweighted pair-group method with the arithmetic averages (UPGMA) clustering algorithm, principal coordinate analysis (PCA) and the Nei's gene diversity index. The studied set of populations split into three main groups based on the AFLP polymorphism analysis. The first group contained L. sativa (control). The second group comprised two L. serriola accessions; one of them was identified as L. serriola f. integrifolia and the other as a mixture of two L. serriola forms. The largest and the most diverse third group contained the remaining L. serriola accessions. The population clustering corresponded approximately to their geographical distribution in Europe. At least five distinct geographic groups were recognised: 1) Northern European; 2) Slovenian; 3) very heterogeneous Central and Western European (mostly north of the Alps); 4) Mediterranean; 5) prevalence of L. serriola f. integrifolia, mostly comprising accessions from the United Kingdom and the Netherlands. This study showed that accessions originating in various eco-geographical conditions of Europe differ significantly in their genetic and protein polymorphism, as well as in morphology. Some European L. serriola populations (e.g. from Scandinavia and United Kingdom/British Isles/) seems to be isolated and homogeneous; in contrast, populations occurring in Central Europe are very diverse and genetically overlapping.     

The p53 Tumor Suppressor Causes Congenital Malformations in Rpl24-Deficient Mice and Promotes Their Survival

Hypomorphic mutation in one allele of ribosomal protein l24 gene (Rpl24) is responsible for the Belly Spot and Tail (Bst) mouse, which suffers from defects of the eye, skeleton, and coat pigmentation. It has been hypothesized that these pathological manifestations result exclusively from faulty protein synthesis. We demonstrate here that upregulation of the p53 tumor suppressor during the restricted period of embryonic development significantly contributes to the Bst phenotype. However, in the absence of p53 a large majority of Rpl24^Bst/+ embryos die. We showed that p53 promotes survival of these mice via p21-dependent mechanism. Our results imply that activation of a p53-dependent checkpoint mechanism in response to various ribosomal protein deficiencies might also play a role in the pathogenesis of congenital malformations in humans.Nascent ribosome biogenesis is required during cell growth, proliferation and differentiation (42, 47). It is temporally and spatially organized within the nucleolus, where rRNAs are transcribed, processed, modified, and assembled with ribosomal proteins (RPS) to generate the mature 40S and 60S ribosomal subunits (13). RPS participate in additional steps in ribosome biogenesis in the nucleoplasm and the cytoplasm, such as the transport of ribosomal precursors, stabilization of ribosome structure, and regulation of different steps in protein synthesis (15).The critical role of at least some RPS in mammals is underscored by the pathological or lethal consequences of the deficiency of just one allele. Only a few heterozygous mutations of RP genes have been shown to be viable in mammals, and each of them has shown a relatively specific phenotype. Germ line heterozygous mutation for RPS19, RPS24, RPS17, RPL35a, RPL5, and RPL11 genes have been found in patients with Diamond-Blackfan anemia, which is characterized by absent or decreased erythropoiesis, and less frequently by small stature and various somatic malformations that mostly occur in the cephalic region, as well as an increased incidence of leukemia, osteogenic sarcoma, and myelodisplastic syndrome (7, 9, 12, 17, 18). Recently, a link between heterozygous mutations in Rps19 and Rps20 and dark skin phenotype in mice has been demonstrated (29).Another heterozygous RP mutant is the Belly Spot and Tail (Bst) mouse (34). This is a semidominant, hypomorphic mutation caused by an intronic deletion in the Rpl24 gene, affecting Rpl24 mRNA splicing. Rpl24^Bst/+ mice are characterized by reduced body size, a white ventral middle spot, white hind feet, retinal abnormalities, a kinked tail, and other skeletal abnormalities. Since Rpl24^Bst/+ mouse embryonic fibroblasts from these mice showed a significant reduction in the rate of overall protein synthesis, it has been suggested that their phenotype result exclusively from faulty translation of mRNAs in tissues that depend on rapid and flawless protein synthesis (34).It has been argued that the differential phenotypes of heterozygous mutants of RP genes in mammals might be attributable to the expression levels of the respective RP and the consequent decrease in the amount of ribosomes, impairment of specific steps in protein synthesis, and potential extraribosomal function. However, it should be pointed out that the relative contribution of the impaired protein synthesis or extraribosomal function to these phenotypes remains to be determined (9, 17, 34, 36, 37, 43, 57).Recent evidence indicates that deficiencies in individual RPS could lead to pathological consequences via activation of a p53-dependent checkpoint regulatory mechanism. We demonstrated that inducible deletion of the Rps6 gene in the liver of adult mice inhibits the synthesis of the 40S ribosomal subunit, as well as proliferation of liver cells, after partial hepatectomy, despite seemingly unaffected protein synthesis (54). These observations suggested the existence of a novel checkpoint, downstream of the deficiency in ribosome biogenesis. Likewise, the perigastrulation lethality of Rps6 heterozygote embryos appears to reflect the triggering of a p53-dependent checkpoint response rather than a deficit in protein synthesis (37). We have assumed that activation of a p53-dependent checkpoint is triggered by impaired rRNA processing in the nucleolus in Rps6-deficient cells, since the nucleolar structure and function are compromised by almost all known p53-inducing stresses (37, 41, 50). Based on all of these observations, it could be speculated that the rare occurrence of RP heterozygosity in mammals reflects the fatal consequences of p53-dependent checkpoint activation (36, 37). In addition to function in development, this checkpoint may also play a role in other processes. Since various RP deficiencies in Drosophila melanogaster, zebrafish, and humans pose a great risk for development of malignant tumors, it is possible that induction of a p53-dependent checkpoint response prevents expansion of such potentially hazardous cells (1, 9, 11, 56).Recently, we initiated an RNA interference screen for RP deficiencies that upregulate the p53 tumor suppressor in A549 cells. It has been previously suggested that a defect in ribosome biogenesis in the nucleolus caused by a RP deficiency triggers the p53 response (36, 37, 50). A number of studies in yeast showed that Rpl24 does not participate in ribosome biogenesis in the nucleolus, but it assembles late with the nascent 60S ribosomes in the cytoplasm and regulates the 60S subunit joining step during translation initiation and other steps in protein synthesis (10, 25, 45). Thus, it was surprising to observe that RPL24 deficiency triggered the p53 response in our screen. This observation led us to consider the possibility that p53 is upregulated in Rpl24^Bst/+ mice. In contrast to previous opinion that the phenotype of these mice results exclusively from impaired protein synthesis (34), we demonstrate here that it is largely caused by the aberrant upregulation of p53 protein expression during embryonic development.     

Comparison of aragonitic molluscan shell proteins

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.     

Immunohistochemical analysis of differentiation in static and mixed prostate cancer spheroids

Neoplastic multicellular spheroids are in vitro models of solid tumors employed in drug testing and basic research. This study compares differentiation in static and mixed prostate cancer spheroids. Staining intensity of prostate specific antigen (PSA) was down-regulated upon mixing from 0.21 ± 0.03 to 0.13 ± 0.03 in LNCaP multicellular spheroids, and from 0.13 ± 0.04 to 0.03 ± 0.02 in DU 145 multicellular spheroids. This was accompanied by 65% increase in the expression of cytokeratins 8 and 18 in DU 145 spheroids. PSA expression extended 60 μm within static spheroids and was disrupted in mixed culture. Diminished PSA expression and spatial organization suggests a more aggressive cancer. Higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation. THus, the existing paradigm of differentiation established for normal tissue does not apply for our neoplastic spheroids. Cell dedifferentiation is attributed to improved interstitial transport and synthesis of extracellular matrix.