A Single Amino Acid Change in the Hemagglutinin Protein of Measles Virus Determines Its Ability To Bind CD46 and Reveals Another Receptor on Marmoset B Cells

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.     

Expressed sequence tag analysis in tef (Eragrostis tef (Zucc) Trotter).

Tef (Eragrostis tef (Zucc.) Trotter) is the most important cereal crop in Ethiopia; however, there is very little DNA sequence information available for this species. Expressed sequence tags (ESTs) were generated from 4 cDNA libraries: seedling leaf, seedling root, and inflorescence of E. tef and seedling leaf of Eragrostis pilosa, a wild relative of E. tef. Clustering of 3603 sequences produced 530 clusters and 1890 singletons, resulting in 2420 tef unigenes. Approximately 3/4 of tef unigenes matched protein or nucleotide sequences in public databases. Annotation of unigenes associated 68% of the putative tef genes with gene ontology categories. Identification of the translated unigenes for conserved protein domains revealed 389 protein family domains (Pfam), the most frequent of which was protein kinase. A total of 170 ESTs containing simple sequence repeats (EST-SSRs) were identified and 80 EST-SSR markers were developed. In addition, 19 single-nucleotide polymorphism (SNP) and (or) insertion-deletion (indel) and 34 intron fragment length polymorphism (IFLP) markers were developed. The EST database and molecular markers generated in this study will be valuable resources for further tef genetic research.     

The Enchytraeid fauna of North Africa

As part of a survey of the Mediterranean enchytraeids, large numbers of samples were collected from a wide range of habitats at 38 localities of Tunisia and Algeria in 1989. Twenty species were identified belonging to six genera. Five species are new records for Tunisia and 17 are new for Algeria. Most of the recorded species are widely distributed throughout Europe, or even cosmopolitan, but some have a more restricted range. Fridericia caprensis has a circummediterranean distribution, Fridericia sardorum seems to be endemic to the western Mediterranean and Fridericia berninii is known only from the Balearic Islands and Algeria. Enchytraeus minutus bisetosus n. ssp. is described. A critical revision of some species is also provided.     

Separation and partial characterization of a type-specific antigen from Chlamydia trachomatis.

Type-specific antigens of Chlamydia trachomatis have been demonstrated by the mouse toxicity prevention test and a variety of immunofluorescent techniques. In addition, biologic activity has been associated with these antigens in terms of type-specific immunity to trachoma infections. This report is the first to describe the detection of a soluble type-specific antigen of C. trachomatis and its separation from those antigens that cross-react among different immunotypes. Test antigens were prepared by labeling the surface components of purified, yolk sac grown organisms with a radioiodinated intermediate (Bolton-Hunter reagent, 125I). The organisms were solubilized with Triton X-100 and gel filtered through Sepharose 6B. All fractions were then tested in radioimmunoassay for binding with rabbit antisera raised against solubilized immunogens prepared from homologous and heterologous strain organisms propagated in BHK-21 cells. A fraction demonstrating homologous binding only was used in subsequent modified procedures for the preparation of quantities of type-specific antigen sufficient for analysis. The antigen appears to be a heat labile, cell surface protein associated with apparent immunogenic activity during the course of actual chlamydial eye infection.     

Chlamydia trachomatis in cell culture. II. Susceptibility of seven established mammalian cell types in vitro. Adaptation of trachoma organisms to McCoy and BHK-21 cells

Trachoma organisms of serotype B were grown serially in irradiated cells (McCoy, BHK-21, Microbiological Associates, and BHK-21, Lister) and tested for infectivity in monolayers of five mammalian cell lines (BHK-21, CHO, HeLa S3, McCoy and OWMK) and two diploid strains (ST/BTL and WI-38). All cell types had low susceptibility to chlamydial infection but the number of inclusions increased when the inoculum was centrifuged onto the monolayers, or when the cells were irradiated. Infection was higher in non-irradiated CHO than in irradiated CHO in three out of a total of six experiments. Inclusion number was increased 300 times in HeLa S3 and up to three times in the other cell types after treatment with diethylaminoethyl-dextran (DEAE-D). Serial passage of Chlamydia trachomatis serotype B (strain Har-36) in CO60 McCoy and CO60 BHK-21 Lister resulted in partial adaptation of the strain to the host cell. The phenomenon of adaptation of serotype B to McCoy compensated for the lower susceptibility of this cell revealed when McCoy cells were inoculated with trachoma elementary bodies grown in BHK-21 Lister or in chick embryo yolk sac. Trachoma organisms of immunotypes A, B and C prepared in yolk sac produced more inclusion-forming units per ml in CO60 BHK-21 Lister than in CO60 McCoy.     

Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a approximately 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.     

Molecular biology of Hendra and Nipah viruses

The structure and genetic organization of Hendra and Nipah viruses places them in the subfamily Paramyxovirinae. However, low homology with other subfamily members and several novel biological and molecular features such as genome length and F(0 )cleavage site suggest classification in a new genus within the Paramyxovirinae.     

Effect of alpha-tocopherol and N-acetylcysteine on benzoyl peroxide toxicity in human keratinocytes

Benzoyl peroxide is a free-radical generating compound widely used in the polymer industry and also in pharmaceuticals as antimicrobial agent to treat acne. However, benzoyl peroxide causes irritation and contact dermatitis in about 1% of patients. Concern over the use of this compound is motivated by the demonstration that it can also act as skin tumor promoter in mice. In addition, benzoyl peroxide induces DNA strand breaks in many cells, including keratinocytes. Benzoyl peroxide toxicity is presumably mediated by the formation of reactive free radicals and by the consumption of intracellular antioxidants.In this work we investigated the effect of both the lipophilic antioxidant alpha-tocopherol and the hydrophilic thiol donor N-acetylcysteine (NAC) in human keratinocyte line HaCaT exposed to benzoyl peroxide. A protective effect against benzoyl peroxide cytotoxicity was achieved when cells were grown on a alpha-tocopherol layer. On the contrary, the addition of alpha-tocopherol dissolved in ethanol had a pro-oxidant effect, leading to an enhancement of benzoyl peroxide toxicity. Cytotoxicity was also reduced adding NAC to the culture medium; the presence of both NAC and alpha-tocopherol exerts a synergistic cytoprotection.     

Preventing microalbuminuria with benazepril,valsartan, and benazepril–valsartan combination therapy in diabetic patients with high-normal albuminuria: A prospective,randomized, open-label,blinded endpoint (PROBE) study

BackgroundAngiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) prevent microalbuminuria in normoalbuminuric type 2 diabetic patients. We assessed whether combined therapy with the 2 medications may prevent microalbuminuria better than ACE inhibitor or ARB monotherapy.Methods and findingsVARIETY was a prospective, randomized, open-label, blinded endpoint (PROBE) trial evaluating whether, at similar blood pressure (BP) control, combined therapy with benazepril (10 mg/day) and valsartan (160 mg/day) would prevent microalbuminuria more effectively than benazepril (20 mg/day) or valsartan (320 mg/day) monotherapy in 612 type 2 diabetic patients with high-normal albuminuria included between July 2007 and April 2013 by the Istituto di Ricerche Farmacologiche Mario Negri IRCCS and 8 diabetology or nephrology units in Italy. Time to progression to microalbuminuria was the primary outcome. Analyses were intention to treat. Baseline characteristics were similar among groups. During a median [interquartile range, IQR] follow-up of 66 [42 to 83] months, 53 patients (27.0%) on combination therapy, 57 (28.1%) on benazepril, and 64 (31.8%) on valsartan reached microalbuminuria. Using an accelerated failure time model, the estimated acceleration factors were 1.410 (95% CI: 0.806 to 2.467, P = 0.229) for benazepril compared to combination therapy, 0.799 (95% CI: 0.422 to 1.514, P = 0.492) for benazepril compared to valsartan, and 1.665 (95% CI: 1.007 to 2.746, P = 0.047) for valsartan compared to combination therapy. Between-group differences in estimated acceleration factors were nonsignificant after adjustment for predefined confounders. BP control was similar across groups. All treatments were safe and tolerated well, with a slight excess of hyperkalemia and hypotension in the combination therapy group. The main study limitation was the lower than expected albuminuria at inclusion.ConclusionsRisk/benefit profile of study treatments was similar. Dual renin–angiotensin system (RAS) blockade is not recommended as compared to benazepril or valsartan monotherapy for prevention of microalbuminuria in normoalbuminuric type 2 diabetic patients.Trial registrationEudraCT 2006-005954-62; ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00503152","term_id":"NCT00503152"}}NCT00503152.

Piero Ruggenenti and co-workers study prevention of microalbuminuria in patients with type 2 diabetes.     

Cryptic diversity among the achaetous Marionina (Annelida,Clitellata, Enchytraeidae)

This study investigates the diversity and taxonomy of a mainly marine group of species lacking chaetae currently assigned to the genus Marionina. This achaetous group includes four nominal species: M. achaeta (Hagen, 1954 Hagen, G. 1954. Michaelsena achaeta nov. sp., ein neuer mariner Oligochaet aus der Kieler Bucht. Faunistische Mitteilungen aus Norddeutschland, 1: 12–13.  [Google Scholar]), M. achaeta sensu Lasserre, 1964 Lasserre, P. 1964. Notes sur quelques oligochètes Enchytraeidae présents dans les plages du Bassin d’Arcachon. Procés-Verbaux des Séances de la Société Linnéenne de Bordeaux, 101: 87–91.  [Google Scholar], M. nevisensis Righi & Kanner, 1979 Righi, G. and Kanner, E. 1979. Marine Oligochaeta (Tubificidae and Enchytraeidae) from the Caribbean Sea. Studies of the Fauna of Curaçao and other Caribbean Islands, 58: 44–68.  [Google Scholar] and M. arenaria Healy, 1979 Healy, B. 1979a. Marine fauna of County Wexford. 1 – Littoral and brackishwater Oligochaeta. The Irish Naturalists' Journal, 19: 418–422.  [Google Scholar]. As Lasserre's (1964 Lasserre, P. 1964. Notes sur quelques oligochètes Enchytraeidae présents dans les plages du Bassin d’Arcachon. Procés-Verbaux des Séances de la Société Linnéenne de Bordeaux, 101: 87–91.  [Google Scholar]) M. achaeta appears to be morphologically different from its (then) senior homonym M. achaeta (Hagen, 1954 Hagen, G. 1954. Michaelsena achaeta nov. sp., ein neuer mariner Oligochaet aus der Kieler Bucht. Faunistische Mitteilungen aus Norddeutschland, 1: 12–13.  [Google Scholar]), the replacement name M. nothachaeta nom. nov. is proposed for it. We studied the genetic and morphological diversity of achaetous specimens of Marionina collected in Florida, the Great Barrier Reef, New Caledonia, Sweden, England and the Bahamas. The collection localities are almost all supralittoral and often brackish-water habitats. Parts of the mitochondrial genes 12S, 16S, COI and the nuclear genes 18S, 28S and ITS were analysed to assess the genetic variation and phylogeny of the achaetous Marionina species. The molecular data reveal one monophyletic group of 11 separately evolving lineages, and between these lineages, K2P distances in the barcoding gene COI vary between 5.4 and 25.0%. On a morphological basis, the lineages could be assigned to seven different groups (morphotypes), of which only two could be identified as described nominal taxa: M. nevisensis s. lat. (several lineages) and M. nothachaeta. Since the former taxon appears to be a complex of cryptic species around the world and the original type material no longer exists, a neotype from the Caribbean was designated for M. nevisensis s. str. The remaining achaetous lineages represent five morphologically distinct species that are left unnamed, awaiting finer morphological scrutiny and detailed comparisons with new collections of M. achaeta and M. arenaria. Summing up, the group of achaetous Marionina now seems to contain up to 13 different species, seven of which are yet to be formally described and named.