Subfamilies of CR1 non-LTR retrotransposons have different 5'UTR sequences but are otherwise conserved

CR1 elements and CR1-related (CR1-like) elements are a novel family of non-LTR retrotransposons that are found in all vertebrates (reptilia, amphibia, fish, and mammals), whereas more distantly related elements are found in several invertebrate species. CR1 elements have several features that distinguish them from other non-LTR retrotransposons. Most notably, their 3' termini lack a polyadenylic acid (poly A) tail and instead contain 2-4 copies of a unique 8 bp repeat. CR1 elements are present at approximately 100,000 copies in the chicken genome. The vast majority of these elements are severely 5' truncated and mutated; however, six subfamilies (CR1-A through CR1-F) are resolved by sequence comparisons. One of these subfamilies (i.e. CR1-B) previously was analyzed in detail. In the present study, we identified several full-length elements from the CR1-F subfamily. Although regions within the open reading frames and 3' untranslated regions of CR1-F and CR1-B elements are well conserved, their respective 5' untranslated regions are unrelated. Thus, our results suggest that new CR1 subfamilies form when elements with intact open reading frames acquire new 5' UTRs, which could, in principle, function as promoters.     

HIV Shedding from Male Circumcision Wounds in HIV-Infected Men: A Prospective Cohort Study

BackgroundA randomized trial of voluntary medical male circumcision (MC) of HIV—infected men reported increased HIV transmission to female partners among men who resumed sexual intercourse prior to wound healing. We conducted a prospective observational study to assess penile HIV shedding after MC.ConclusionPenile HIV shedding is significantly reduced after healing of MC wounds. Lower plasma VL is associated with decreased frequency and quantity of HIV shedding from MC wounds. Starting ART prior to MC should be considered to reduce male-to-female HIV transmission risk. Research is needed to assess the time on ART required to decrease shedding, and the acceptability and feasibility of initiating ART at the time of MC.     

Posttranslational processing of human lysosomal acid beta-glucosidase: a continuum of defects in Gaucher disease type 1 and type 2 fibroblasts.

The major processing steps in the maturation of the lysosomal hydrolase, acid beta-glucosidase, were examined in fibroblasts from normal individuals and from patients with types 1 and 2 Gaucher disease. In pulse-chase studies with normal fibroblasts, remodeling of N-linked oligosaccharides resulted in the temporal appearance of three molecular-weight forms of acid beta-glucosidase. An initial 64-kDa form, containing high mannose-type oligosaccharide side chains, was processed quantitatively, within 24 h, to a sialylated 69-kDa form. During the subsequent 96 h, some of the 69-kDa form is processed to 59 kDa. Glycosidase digestion studies revealed that the increase in the apparent molecular weight of the normal enzyme from 64 kDa to 69 kDa resulted primarily from the addition to sialic acid residues in the Golgi apparatus. The polypeptide backbone of both the 64-kDa and 69-kDa forms was 55.3 kDa. Processing of acid beta-glucosidase in fibroblasts from three of four type 1 (nonneuronopathic) Ashkenazi Jewish Gaucher disease patients was nearly normal. With fibroblasts from one Ashkenazi Jewish and three non-Jewish type 1 as well as from two type 2 (acute neuronopathic) Gaucher disease patients, only a 64-kDa form of acid beta-glucosidase was detected. Inefficient and incomplete processing to the 69-kDa form was found in one type 2 cell line (GM2627). These results indicate that no firm correlation exists between the type or degree of abnormal processing of acid beta-glucosidase in fibroblasts and the phenotype of Gaucher disease.     

Fluorescence probes in biochemistry: an examination of the non-fluorescent behavior of dansylamide by photoacoustic calorimetry.

Photoacoustic calorimetry is shown to be a simple, precise, and accurate method for the quantification of the photophysics of a fluorescence probe, e.g., dansylamide, in a variety of solvents. This technique, which is described in detail, provides a direct measurement of the energy that is released nonradiatively following photostimulation, and can therefore be used to indirectly determine the amount of energy released via luminescent pathways. Photoacoustic calorimetry combined with established absorption and fluorescence methodologies provides a complete arsenal for characterizing the photophysical properties of many systems. Comparison of the photoacoustic signal for dansylamide versus standard compounds (ferrocene, tetraphenylethylene, 8-anilinonaphthalene-1-sulfonate, and/or 5,5'-dithiobis(2-nitrobenzoic acid) in 12 different solvents gave fh values (fraction of each absorbed 337.1-nm photon returned as heat) from a low of 0.530 in 1,4-dioxane to a high of 0.973 in water. The trend noted with solvent polarity is different and more revealing than that determined by the more classical approach of examining either the wavelength of the emission maximum or the fluorescence quantum yield.     

Serotyping of <Emphasis Type="Italic">Proteus mirabilis</Emphasis> clinical strains based on lipopolysaccharide O-polysaccharide and core oligosaccharide structures

The aim of this work was to serotype Proteus mirabilis urinary tract infection (UTI) strains based on chemically defined O-antigens with the use of two clinical collections from Sweden and Poland consisting of 99 and 24 UTI strains, respectively. A simple two-step serotyping scheme was proposed using enzyme immunoassay with heat-stable surface antigens of Proteus cells and immunoblotting with isolated lipopolysaccharides (LPSs). Using polyclonal anti-P. mirabilis rabbit antisera, 50 Swedish and 8 Polish strains were classified into serogroups O10, O38, O36, O30, O17, O23, O9, O40, O49, O27, O5, O13, O24, O14, and O33. From the Swedish strains, 10 belonged to serogroup O10 and five to each of serogroups O38, O36, and O9. Therefore, none of the O-serogroups was predominant. The majority of the serotyped clinical strains possess acidic O-antigens containing uronic acids and various acidic non-carbohydrate substituents. In immunoblotting, antisera cross-reacted with both O-antigen and core of LPSs. The core region of 19 LPSs bound a single serum, and that of 12 LPSs bound more than two sera. Following bioinformatic analysis of the available sequences, a molecular approach to the prediction of Proteus core oligosaccharide structures was proposed. The identification of the core type of P. mirabilis R110, derived from a serogroup O3 wild strain, using restriction fragments length polymorphism analysis of galacturonic acid transferase is shown as an example. In summary, the most frequent O-serogroups among P. mirabilis UTI stains were identified. The diversity of serological reactions of LPSs is useful for serotyping of P. mirabilis clinical isolates. A possible role of the acidic components of O-antigens in UTI is discussed.     

Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation

Key message

Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent.

Abstract

Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein’s N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.     

Edge effects influence the composition and density of reef residents on subtidal restored oyster reefs

Within estuarine and coastal ecosystems globally, extensive habitat degradation and loss threaten critical ecosystem functions and necessitate widescale restoration efforts. There is abundant evidence that ecological processes and species interactions can vary with habitat characteristics, which has important implications for the design and implementation of restoration efforts aimed at enhancing specific ecosystem functions and services. We conducted an experiment examining how habitat characteristics (presence; edge vs. interior) influence the communities of resident fish and mobile invertebrates on restored oyster (Crassostrea virginica) reefs. Similar to previous studies, we found that restored reefs altered community composition and augmented total abundance and biomass relative to unstructured sand habitat. Community composition and biomass also differed between the edge and interior of individual reefs as a result of species-specific patterns over small spatial scales. These patterns were only weakly linked to oyster density, suggesting that other factors that vary between edge and interior (e.g. predator access or species interactions) are likely more important for community structure on oyster reefs. Fine-scale information on resident species' use of oyster reefs will help facilitate restoration by allowing decision makers to optimize the amount of edge versus interior habitat. To improve the prediction of faunal use and benefits from habitat restoration, we recommend investigations into the mechanisms shaping edge and interior preferences on oyster reefs.     

Evidence of phenotypic plasticity and local adaption in metabolic rates between components of the Icelandic cod (Gadus morhua L.) stock

Phenotypic plasticity and local adaptations are important considerations in delineating population structure of marine fishes and critical to their conservation and management. We compared the weight-specific oxygen consumption rates (VO₂/M) of juvenile cod from the northern and southern components of the Icelandic stock acclimated to 4.0°C, 8.5°C, and 12.6°C and their metabolic response to abrupt temperatures changes within this range. Southern individuals exhibited VO₂/M up to 50% higher than their northern counterparts when tested at their acclimation temperature. However, northern fish generally experienced greater changes in VO₂/M, three to six-fold increases, relative to that expected at acclimation when moved to higher temperatures. Southern cod showed a greater decrease in VO₂/M when exposed to lower temperatures. Our results indicate physiological differences exist between the northern and southern components of the Icelandic cod stock and warrant considering them as two distinct populations.     

High frequency of the Gaucher disease mutation at nucleotide 1226 among Ashkenazi Jews.

Reliable estimates of the frequency of Gaucher disease-producing mutations are not available. The high frequency of Gaucher disease in the Ashkenazi Jewish population is due to the occurrence of a mutation at nucleotide (nt) 1226. We have screened 593 DNA samples from normal Ashkenazi Jews, as well as 62 DNA samples from all our Ashkenazi Jewish patients with Gaucher disease, for the presence of the 1226 mutation. In the 593 presumed normal Ashkenazi Jewish individuals the 1226 mutation was identified in the heterozygous state in 37 and in the homozygous state in two, giving a gene frequency of .035 for the mutation. This 1226 mutation represented 73% of the 124 Gaucher disease alleles in Jewish Gaucher disease patients. Accordingly we estimate that the gene frequency for Gaucher disease among the Ashkenazi Jewish population is .047, which is equivalent to a carrier frequency of 8.9% and a birth incidence of 1:450.