Exposure to Silver Nanospheres Leads to Altered Respiratory Mechanics and Delayed Immune Response in an in Vivo Murine Model.

Here we examine the organ level toxicology of both carbon black (CB) and silver nanoparticles (AgNP). We aim to determine metal-specific effects to respiratory function, inflammation and potential interactions with lung lining fluid (LLF). C57Bl6/J male mice were intratracheally instilled with saline (control), low (0.05 µg/g) or high (0.5 µg/g) doses of either AgNP or CB 15 nm nanospheres. Lung histology, cytology, surfactant composition and function, inflammatory gene expression, and pulmonary function were measured at 1, 3, and 7 days post-exposure. Acutely, high dose CB resulted in an inflammatory response, increased neutrophilia and cytokine production, without alteration in surfactant composition or respiratory mechanics. Low dose CB had no effect. Neither low nor high dose AgNPs resulted in an acute inflammatory response, but there was an increase in work of breathing. Three days post-exposure with CB, a persistent neutrophilia was noted. High dose AgNP resulted in an elevated number of macrophages and invasion of lymphocytes. Additionally, AgNP treated mice displayed increased expression of IL1B, IL6, CCL2, and IL10. However, there were no significant changes in respiratory mechanics. At day 7, inflammation had resolved in AgNP-treated mice, but tissue stiffness and resistance were significantly decreased, which was accompanied by an increase in surfactant protein D (SP-D) content. These data demonstrate that the presence of metal alters the response of the lung to nanoparticle exposure. AgNP-surfactant interactions may alter respiratory function and result in a delayed immune response, potentially due to modified airway epithelial cell function.

The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening.

BACKGROUND: Cystic fibrosis (CF) is caused by compound heterozygosity or homozygosity of CF transmembrane conductance regulator gene (CFTR) mutations. Phenotypic variability associated with certain mutations makes genetic counselling difficult, notably for R117H, whose disease phenotype varies from asymptomatic to classical CF. The high frequency of R117H observed in CF newborn screening has also introduced diagnostic dilemmas. The aim of this study was to evaluate the disease penetrance for R117H in order to improve clinical practice. METHODS: The phenotypes in all individuals identified in France as compound heterozygous for R117H and F508del, the most frequent CF mutation, were described. The allelic prevalences of R117H (p(R117H)), on either intron 8 T5 or T7 background, and F508del (p(F508del)) were determined in the French population, to permit an evaluation of the penetrance of CF for the [R117H]+[F508del] genotype. RESULTS: Clinical details were documented for 184 [R117H]+[F508del] individuals, including 72 newborns. The disease phenotype was predominantly mild; one child had classical CF, and three adults' severe pulmonary symptoms. In 5245 healthy adults, p(F508del) was 1.06%, p(R117H;T7) 0.27% and p(R117H;T5)<0.01%. The theoretical number of [R117H;T7]+[F508del] individuals in the French population was estimated at 3650, whereas only 112 were known with CF related symptoms (3.1%). The penetrance of classical CF for [R117H;T7]+[F508del] was estimated at 0.03% and that of severe CF in adulthood at 0.06%. CONCLUSIONS: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes. The real impact of so-called disease mutations should be assessed before including them in newborn or preconceptional carrier screening programmes.

A modified ELISA and western blot accurately determine anti-human immunodeficiency virus type 1 antibodies in oral fluids obtained with a special collecting device.

Serum and saliva from 195 known human immunodeficiency virus (HIV)-seropositive patients and 198 military personnel undergoing annual HIV serologic testing were evaluated in a prospective, blinded fashion for anti-HIV-1 antibodies. Oral specimens, collected with a device designed to concentrate oral mucosal transudate from whole saliva, were tested by a modified ELISA and by Western blot. Serum was tested in a standard manner. All 195 HIV-1-seropositive subjects had detectable anti-HIV-1 antibodies in their saliva by ELISA; 190 saliva samples were positive by Western blot and 5 were indeterminate. None of the 198 military personnel were positive by ELISA of serum or oral fluid. The sensitivity, specificity, and positive and negative predictive values for ELISA of saliva were each 100%. The serologic testing of oral mucosal transudate appears to be a simple, safe, sensitive, and specific method for detecting anti-HIV-1 antibodies.

Phylogenic relationships of the amino acid sequences of prosome (proteasome, MCP) subunits.

Prosomes [or proteasomes, Multi-Catalytic Proteinase (MCP) are multisubunit protein complexes, found from archaebacteria to man, the structure of which (a 4-layer cylinder) is remarkable conserved. They were first observed as subcomplexes of untranslated mRNP, and then as a multicatalytic proteinase with several proteolytic activities. A number of sequences from subunits of these complexes are now available. Analysis of the sequences shows that these subunits are evolutionarily related, and reveals three highly conserved amino acid stretches. Based on a phylogenic approach, we propose to classify the sequenced subunits into 14 families, which fall into two superfamilies, of the alpha- and beta-type. These data, together with several recently published observations, suggest that some subunits may be interchangeable within the complexes, which would thus constitute a population of heterogenous particles.

The prosomal RNA-binding protein p27K is a member of the alpha-type human prosomal gene family.

Monoclonal antibodies demonstrated high conservation during evolution of a prosomal protein of M(r) 27,000 and differentiation--specific expression of the epitope. More than 90% of the reacting antigen was found as a p27K protein in the free messenger ribonucleoprotein (mRNP) fraction but another protein of M(r) 38,000, which shared protease fingerprint patterns with the p27K polypeptide, was also labelled in the nuclear and polyribosomal fractions. Sequencing of cDNA recombinant clones encoding the p27/38K protein and comparison with another prosomal protein, p30-33K, demonstrated the existence of a common characteristic sequence pattern containing three highly conserved segments. The genes Hs PROS-27 and Hs PROS-30 were mapped to chromosomes 14 (14q13) and 11 (11p15.1), respectively. The structure of the p27K protein shows multiple potential phosphorylation sites, an NTP-binding fold and an RNA-binding consensus sequence. The Hs PROS-27/beta-galactosidase fusion protein binds a single RNA of about 120 nucleotides from total HeLa cell RNA. Sequence comparisons show that the Hs PROS-27 and Hs PROS-30 genes belong to the gene family that encodes the prosome--MCP (multicatalytic proteinase)--proteasome proteins. Comparison with other members of the family from various species allowed us to show that the tripartite consensus sequence characteristic of the alpha-type sub-family is conserved from archeobacteria to man. The members of this gene family are characterised by very high evolutionary conservation of amino acid sequences of homologous genes and 20%-35% sequence similarity, between different family member within the same species and are clearly distinct from the beta-type family.

Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA.

Prosomes are ribonucleoprotein particles constituted by a variable set of about 20 proteins found associated with untranslated mRNA. In addition, they contain a small RNA, the presence of which has been an issue of controversy for a long time. The intact particles have a multicatalytic proteinase (MCP) activity and are very stable; we have never observed autodigestion of the particle by its intrinsic proteinase activity. Surprisingly it was found that Zn2+ and Cu2+ ions at concentrations of 0.1-1 mM disrupt the prosome particles isolated from HeLa cells and duck erythroblasts and abolish instantaneously its MCP activity, without altering the two-dimensional electrophoretic pattern of the constituent proteins. Fe2+, however, seems to induce autodegradation rather than dissociation of the prosome constituents. Most interestingly, protein or oligopeptide substrates protect the particle and its proteinase activity from disruption by Zn2+ or Cu2+. Nuclease-digestion assays reveal that the prosomal RNA, which is largely resistant in the intact particle, becomes digestible after dissociation of prosomes by Zn2+. These data give, for the first time, unambiguous proof of the presence of an RNA in the particle. Furthermore, they demonstrate a structure-function relationship between the complex and its enzyme activity, which seems to be based on the particle as an entity and not on the single constituent proteins.

Molecular cloning of cDNAs for rat proteasomes: deduced primary structures of four other subunits.

Proteasomes (multi-protease complexes) are composed of approximately 15 non-identical subunits of similar sizes (molecular weight = 21-32 kDa), but different charges (isoelectric point = 4-9). Previously, we deduced the primary structures of 6 subunits of rat proteasomes by recombinant DNA techniques. In this paper we report the nucleotide sequences of 4 other subunits, rIOTA, rZETA, rDELTA, and rRING12, determined from cDNA clones isolated by screening a rat H4TG hepatoma cell cDNA library with the cDNAs of their human counterparts as probes. The polypeptides deduced from their nucleotide sequences consisted of 246, 241, 202, and 219 amino acid residues with calculated molecular weights of 27,399, 26,391, 21,649, and 23,324, and calculated isoelectric points of 6.37, 4.65, 4.84, and 4.70, respectively. These results and previous findings indicate that the primary structures of the subunits of rat proteasomes show considerably high inter-subunit homology, but can be classified into apparently distinct sub-groups, suggesting that rat proteasome genes form a multi-gene family with the same evolutionary origin, but have diverged during evolution to acquire possibly subunit-specific functions.

Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes.

Screening of a lambda gt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M(r) of 30,227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosome-specific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95-102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29-38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.

The protein of M(r) 21,000 constituting the prosome-like particle of duck erythroblasts is homologous to apoferritin.

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this '35S' mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23-33]. The PLP is a multimer of a protein of M(r) 21,000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of M(r) 21,000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage.

Prosomes and their multicatalytic proteinase activity.

Prosomes were first described as being mRNA-associated RNP (ribonucleoprotein) particles and subcomponents of repressed mRNPs (messenger ribonucleoprotein). We show here that prosomes isolated from translationally inactive mRNP have a protease activity identical to that described by others for the multicatalytic proteinase complex (MCP, 'proteasome'). By RNase or non-ionic detergent treatment, the MCP activity associated with repressed non-globin mRNP from avian erythroblasts, sedimenting at 35 S, could be quantitatively shifted on sucrose gradients to the 19-S sedimentation zone characteristic of prosomes, which were identified by monoclonal antibodies. The presence of small RNA in the enzymatic complex was shown by immunoprecipitation of the protease activity out of dissociated mRNP using a mixture of anti-prosome monoclonal antibodies; a set of small RNAs 80-120 nucleotides long was isolated from the immunoprecipitate. Furthermore, on CsCl gradients, colocalisation of the MCP activity with prosomal proteins and prosomal RNA was found, and no difference in the prosomal RNA pattern was observed whether the particles were fixed or not prior to centrifugation. These data indicate that the MCP activity is a property of prosomes, shown to be in part RNP and subcomplexes of in vivo untranslated mRNP. A hypothesis for the role of the prosome-MCP particles in maintaining homeostasis of specific protein levels is proposed.