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The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure (Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table (Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.
Open in a separate windown/a, not applicable. Data based on [6]. 相似文献
Table 1
Genome sizes in land plantsLineage | Range (1C; pg) | Mean |
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Gymnosperms | ||
Conifers | ||
Pinaceae | 9.5-36.0 | 23.7 |
Cupressaceae | 8.3-32.1 | 12.8 |
Sciadopitys | 20.8 | n/a |
Gnetales | ||
Ephedraceae | 8.9-15.7 | 8.9 |
Gnetaceae | 2.3-4.0 | 2.3 |
Cycadaceae | 12.6-14.8 | 13.4 |
Ginkgo biloba | 11.75 | n/a |
Monilophytes | ||
Ophioglossaceae | 10.2-65.6 | 31.05 |
Equisetaceae | 12.9-304 | 22.0 |
Psilotum | 72.7 | n/a |
Leptosporangiate ferns | ||
Polypodiaceae | 7.5-19.7 | 7.5 |
Aspleniaceae | 4.1-9.1 | 6.2 |
Athyriaceae | 6.3-9.3 | 7.6 |
Dryopteridaceae | 6.8-23.6 | 11.7 |
Water ferns | ||
Azolla | 0.77 | n/a |
Angiosperms | ||
Oryza sativa | 0.50 | n/a |
Amborella trichopoda | 0.89 | n/a |
Arabidopsis thaliana | 0.16 | n/a |
Zea mays | 2.73 | n/a |
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José Carlos Quintela Francisco García-del Portillo Ernst Pittenauer Günter Allmaier Miguel A. de Pedro 《Journal of bacteriology》1999,181(1):334-337
Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)2 bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear 252californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H+ and Na+ ions and in the negative ion mode with H− and CN− ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. (Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. (Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)2 bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A204 of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.
Open in a separate windowaDR5 and DR10 to DR13 were analyzed in both the positive and negative ion modes. Muropeptides DR1 to DR4 and DR6 to DR9 were analyzed in the positive mode only due to the small amounts of sample available. bMass difference between measured and calculated quasimolecular ion values. c[(Measured mass−calculated mass)/calculated mass] × 100. dN-Acetylglucosamine. eN-Acetylmuramitol. f(1→6)Anhydro-N-acetylmuramic acid. Structural assignments of muropeptides DR1, DR2, DR8 to DR10, and DR12 deserve special comments. The low m/z value measured for DR1 (700.1) fitted very well with the value calculated for GlucNAc–MurNAc–l-Ala–d-Glu (699.69). Even smaller was the mass deduced for DR9 from the m/z value of the molecular ion of the sodium adduct (702.1) (Fig. (Fig.2).2). The mass difference between DR1 and DR9 (19.9 mass units) was very close indeed to the calculated difference between N-acetylmuramitol and the (1→6)anhydro form of MurNAc (20.04 mass units). Therefore, DR9 was identified as GlucNAc–(1→6)anhydro-MurNAc–l-Ala–d-Glu (Fig. (Fig.1).1). Muropeptides with (1→6)anhydro muramic acid have been identified in mureins from diverse origins (10, 15, 17, 19), indicating that it might be a common feature among peptidoglycan-containing microorganisms. Open in a separate windowFIG. 2Positive-ion linear PDMS of muropeptide DR9. Muropeptide DR9 was purified, desalted by HPLC, and subjected to PDMS to determine the molecular mass. The masses for the dominant molecular ions are indicated.The measured m/z value for the [M+Na]+ ion of DR8 was 1,521.6, very close to the mass calculated for a cross-linked dimer without one disaccharide moiety (1,520.53) (Fig. (Fig.1;1; Table Table1).1). Such muropeptides, also identified in T. thermophilus HB8 and other bacteria (18, 19), are most likely generated by the enzymatic clevage of MurNAc–l-Ala amide bonds in murein by an N-acetylmuramyl–l-alanine amidase (21). In particular, DR8 could derive from DR11. The difference between measured m/z values for DR8 and DR11 was 478.7, which fits with the mass contribution of a disaccharide moiety (480.5) within the mass accuracy of the instrument.The m/z values for muropeptides DR2, DR10, and DR12 supported the argument for structures in which the two d-Ala residues from the d-Ala–d-Ala C-terminal dipeptide were lost, leaving Orn as the C-terminal amino acid.The position of one Gly residue in muropeptides DR2, DR8, and DR10 to DR13 could not be formally demonstrated. One of the Gly residues could be at either the N- or the C-terminal positions. However, the N-terminal position seems more likely. The structure of the basic muropeptide (DR7), with a (Gly)2 acylating the δ-NH2 group of Orn, suggests that major muropeptides should present a (Gly)2 dipeptide. The scarcity of DR3 and DR6, which unambiguously have Gly as the C-terminal amino acid (Fig. (Fig.1),1), supports our assumption.Molar proportions for each muropeptide were calculated as proposed by Glauner et al. (10) and are shown in Table Table1.1. For calculations the structures of DR10 to DR13 were assumed to be those shown in Fig. Fig.1.1. The degree of cross-linkage calculated was 47.2%. Trimeric muropeptides were rather abundant (8 mol%) and made a substantial contribution to total cross-linkage. However, higher-order oligomers were not detected, in contrast with other gram-positive bacteria, such as Staphylococcus aureus, which is rich in such oligomers (8). The proportion of muropeptides with (1→6)anhydro-muramic acid (5 mol%) corresponded to a mean glycan strand length of 20 disaccharide units, which is in the range of values published for other bacteria (10, 17).The results of our study indicate that mureins from D. radiodurans and T. thermophilus HB8 (19) are certainly related in their basic structures but have distinct muropeptide compositions. In accordance with the phylogenetic proximity of Thermus and Deinococcus (11), both mureins are built up from the same basic monomeric subunit (DR7 in Fig. Fig.1),1), are cross-linked by (Gly)2 bridges, and have (1→6)anhydro-muramic acid at the termini of glycan strands. Most interestingly, Deinococcus and Thermus are the only microorganisms identified at present with the murein chemotype A3β as defined by Schleifer and Kandler (20). Nevertheless, the differences in muropeptide composition were substantial. Murein from D. radiodurans was poor in d-Ala–d-Ala- and d-Ala–Gly-terminated muropeptides (2.2 and 2.4 mol%, respectively) but abundant in Orn-terminated muropeptides (23.8 mol%) and in muropeptides with a peptide chain reduced to the dipeptide l-Ala–d-Glu (18 mol%). In contrast, neither Orn- nor Glu-terminated muropeptides have been detected in T. thermophilus HB8 murein, which is highly enriched in muropeptides with d-Ala–d-Ala and d-Ala–Gly (19). Furthermore, no traces of phenyl acetate-containing muropeptides, a landmark for T. thermophilus HB8 murein (19), were found in D. radiodurans. Cross-linkage was definitely higher in D. radiodurans than in T. thermophilus HB8 (47.4 and 27%, respectively), largely due to the higher proportion of trimers in the former.The similarity in murein basic structure suggests that the difference between D. radiodurans and T. thermophilus HB8 with respect to the Gram reaction may simply be a consequence of the difference in the thickness of cell walls (2, 3, 23). Interestingly, D. radiodurans murein turned out to be relatively simple for a gram-positive organism, possibly reflecting the primitive nature of this genus as deduced from phylogenetic trees (11). Our results illustrate the phylogenetic proximity between Deinococcus and Thermus at the cell wall level but also point out the structural divergences originated by the evolutionary history of each genus. 相似文献
TABLE 1
Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radioduransMuropeptidea | Ion | m/z
| Δmb | Error (%)c | Muropeptide composition
| Muropeptide abundance (mol%) | ||||||
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Calculated | Measured | NAGd | NAMe | Glu | Orn | Ala | Gly | |||||
DR1 | [M+H]+ | 699.69 | 700.1 | 0.41 | 0.06 | 1 | 1 | 1 | 0 | 1 | 0 | 12.0 |
DR2 | [M+H]+ | 927.94 | 928.3 | 0.36 | 0.04 | 1 | 1 | 1 | 1 | 1 | 2 | 5.7 |
DR3 | [M+Na]+ | 1,006.97 | 1,007.5 | 0.53 | 0.05 | 1 | 1 | 1 | 1 | 1 | 3 | 3.0 |
DR4 | [M+Na]+ | 963.95 | 964.6 | 0.65 | 0.07 | 1 | 1 | 1 | 1 | 2 | 1 | 2.5 |
DR5 | [M+H]+ | 999.02 | 999.8 | 0.78 | 0.08 | 1 | 1 | 1 | 1 | 2 | 2 | 27.7 |
[M−H]− | 997.00 | 997.3 | 0.30 | 0.03 | ||||||||
DR6 | [M+Na]+ | 1,078.5 | 1,078.8 | 0.75 | 0.07 | 1 | 1 | 1 | 1 | 2 | 3 | 2.4 |
DR7 | [M+H]+ | 1,070.09 | 1,071.0 | 0.90 | 0.08 | 1 | 1 | 1 | 1 | 3 | 2 | 2.2 |
DR8 | [M+Na]+ | 1,520.53 | 1,521.6 | 1.08 | 0.07 | 1 | 1 | 2 | 2 | 4 | 4 | 2.2 |
DR9 | [M+Na]+ | 701.64 | 702.1 | 0.46 | 0.03 | 1 | 1f | 1 | 0 | 1 | 0 | 5.0 |
DR10 | [M+H]+ | 1,907.94 | 1,907.8 | 0.14 | 0.01 | 2 | 2 | 2 | 2 | 3 | 4 | 10.1 |
[M−H]− | 1,905.92 | 1,906.6 | 0.68 | 0.04 | ||||||||
DR11 | [M+H]+ | 1,979.01 | 1,979.1 | 0.09 | 0.01 | 2 | 2 | 2 | 2 | 4 | 4 | 19.1 |
[M−H]− | 1,977.00 | 1,977.3 | 0.30 | 0.02 | ||||||||
DR12 | [M+H]+ | 2,887.93 | 2,886.5 | −1.43 | −0.05 | 3 | 3 | 3 | 3 | 5 | 6 | 4.4 |
[M−H]− | 2,885.91 | 2,885.8 | −0.11 | −0.01 | ||||||||
DR13 | [M+H]+ | 2,959.00 | 2,957.8 | −1.20 | −0.04 | 3 | 3 | 3 | 3 | 6 | 6 | 3.6 |
[M−H]− | 2,956.99 | 2,955.9 | −1.09 | −0.04 |
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Catherine A. Blish Zahra Jalalian-Lechak Stephanie Rainwater Minh-An Nguyen Ozge C. Dogan Julie Overbaugh 《Journal of virology》2009,83(15):7783-7788
The human immunodeficiency virus type 1 (HIV-1) variants that are transmitted to newly infected individuals are the primary targets of interventions, such as vaccines and microbicides, aimed at preventing new infections. Newly acquired subtype A, B, and C variants have been the focus of neutralization studies, although many of these viruses, particularly of subtypes A and B, represent viruses circulating more than a decade ago. In order to better represent the global diversity of transmitted HIV-1 variants, an additional 31 sexually transmitted Kenyan HIV-1 env genes, representing several recent infections with subtype A, as well as subtypes A/D, C, and D, were cloned, and their neutralization profiles were characterized. Most env variants were resistant to neutralization by the monoclonal antibodies (MAbs) b12, 4E10, 2F5, and 2G12, suggesting that targeting the epitopes of these MAbs may not be effective against variants that are spreading in areas of endemicity. However, significant cross-subtype neutralization by plasma was observed, indicating that there may be other epitopes, not yet defined by the limited available MAbs, which could be recognized more broadly.Most effective viral vaccines are thought to provide protection primarily by stimulating neutralizing antibodies (NAbs) to clear cell-free virus (25, 27). Because protection by NAbs requires recognition of common viral epitopes, the extreme genetic diversity of human immunodeficiency virus type 1 (HIV-1) presents a particular challenge to NAb-based vaccine approaches. Therefore, a critical starting point for studies of immune-mediated protection against HIV-1 is a collection of newly transmitted HIV-1 variants, particularly from areas of endemicity, such as sub-Saharan Africa, in order to determine whether vaccines are appropriately targeted to common epitopes from these relevant transmitted strains.During HIV-1 transmission, a bottleneck allows only one or a few variants to be transmitted to a newly infected individual (6, 9, 16, 29, 34, 37, 39), and the sensitivity of these early transmitted strains to antibody-mediated neutralization is therefore of particular interest. Newly transmitted HIV-1 variants have demonstrated significant heterogeneity in their neutralization phenotypes both within and between subtypes (2, 3, 6-8, 11, 13-15, 22, 30, 32, 36). Panels of sexually transmitted HIV-1 envelope variants (based on the envelope gene, env) have been characterized, including subtype B variants from North America, Trinidad, and Europe, subtype C variants from South Africa and Zambia, and subtype A variants from Kenya collected between 1994 and 1996 (2, 14, 15). Here, we characterize an additional 31 envelope variants from 14 subjects with sexually transmitted HIV-1 who were infected in Kenya, where subtypes A, C, and D circulate, between 1993 and 2005 (24, 31).The env genes were cloned from samples drawn 14 to 391 (median, 65) days postinfection from individuals enrolled in a prospective cohort of high-risk women in Mombasa, Kenya (19-21). Demographic characteristics of the subjects are summarized in Table Table1;1; the timing of first infection was determined by both HIV-1 serology and HIV RNA testing as described previously (12). All of the subjects were presumably infected by male-to-female transmission and displayed a range of plasma viral loads at the time of env gene cloning (Table (Table1).1). For most individuals, full-length env genes were cloned from uncultured peripheral blood mononuclear cell (PBMC) DNA, though for two individuals, clones were obtained from DNA following short-term coculture with donor PBMCs (Table (Table1).1). env genes were cloned by single-copy nested PCR with primers and PCR conditions as described previously (4, 17). We tested env genes for their ability to mediate infection by transfecting env plasmid DNA into 293T cells along with an env-deficient HIV-1 subtype A proviral plasmid, Q23Δenv, to make pseudoviral particles (17). More than 80 env clones were obtained from 16 subjects; less than one-half were functional on the basis of the infectivity of pseudoviral particles in a single-round infection of TZM-bl cells (AIDS Research and Reference Reagent Program, National Institutes of Health), as observed previously for env genes cloned from proviral sequences (17); a lower fraction of functional env genes have been reported from plasma (18). We focused on the proviral sequences here because they presumably best represent the sequence closest to that of the transmitted strains. The 31 functional env variants are described in Table Table11.
Open in a separate windowadpi, days postinfection as defined by RNA testing (12).bVL, viral load on the sample date in which env genes were cloned.cucPBMC, uncultured PBMCs; ccPBMC, cocultured PBMCs.dAverage pairwise distance between the full-length env variants from a given subject. NA, not applicable because there was only one variant available from the subject.eenv variants from these two subjects were cloned from >6 months postinfection, as noted, and should not be considered true early env variants.The full-length, functional env genes were sequenced and aligned to generate a maximum likelihood phylogenetic tree with reference sequences from the Los Alamos National Laboratory HIV database, as described previously (26). Viral env clones from the same subject clustered together, and a wide spectrum of genetic diversity was observed overall (Fig. (Fig.1).1). Some women, such as subject QF495, were infected with a relatively homogeneous viral population, with average pairwise differences of only 0.12% between env variants (Table (Table11 and Fig. Fig.1).1). However, as observed previously in this cohort (16, 28, 29, 33-35), other individuals, such as subjects QH359 and QD435, were infected with more heterogeneous viral populations with average pairwise differences of 1.4% and 0.88% between variants, respectively (Table (Table11 and Fig. Fig.1).1). env genes from subtypes A (13 variants), C (3 variants), and D (8 variants), as well as A/D recombinants (4 variants) and A2/D recombinants (3 variants), were represented (Fig. (Fig.1).1). The viral subtypes were confirmed using the NCBI genotyping database (http://www.ncbi.nlm.nih.gov/).Open in a separate windowFIG. 1.Maximum likelihood phylogenetic tree of full-length sequences from early subtype A, C, D, and A/D recombinant env variants in Kenya. The 31 novel env clones from Kenyan early infections and reference sequences for subtypes A, B, C, D, and K from the Los Alamos HIV database (http://www.hiv.lanl.gov/content/index) are displayed. The phylogenetic tree was rooted with subtype K env sequences. Values at nodes indicate the percentage of bootstraps in which the cluster the right was found; only values of 70% or greater are shown.The deduced amino acid sequences revealed that all functional variants had an uninterrupted open reading frame in env except for variant QB099.391I.ENV.C8, which had a frameshift mutation within the cytoplasmic tail of gp41. There was significant heterogeneity in the length of the protein variable loops, particularly V1/V2, which ranged from 57 amino acids (aa) to 113 aa (Table (Table1).1). The V3, V4, and V5 loops also varied in length, though less dramatically (Table (Table1).1). Variants from the same subject were generally similar in their variable-loop lengths. Moderate variation was also observed in the number and position of potential N-linked glycosylation sites (PNGS) (Table (Table11).Previous analyses indicated that early subtype C env proteins had shorter variable loops than did early subtype B env proteins (13), suggesting that there are different env protein features between subtypes. Thus, to compare variable-loop lengths and the numbers of PNGS between subtypes using this expanded group of early env variants, we evaluated the 31 newly cloned variants plus an additional 15 subtype A variants (2), 19 subtype B variants (14), and 18 subtype C variants (15) from other early virus panels. In order to avoid bias, when more than one env variant was available from a subject, the average loop length or PNGS number for that subject''s env proteins was used. We did not observe significant differences in V1/V2 length, V5 length, or the numbers of PNGS between subtypes by the Kruskal-Wallis equality-of-populations rank test (Table (Table2)2) . However, there were significant differences between the V3 and V4 loop lengths of the subtypes after adjusting for multiple comparisons (Table (Table2).2). The differences in V3 length appeared to be a result of shorter V3 loops in subtype D env proteins than in early subtype B (P = 0.006) or C (P < 0.001) env proteins (Table (Table2).2). The differences in V4 length were caused by shorter V4 loops in subtype C env proteins in comparison to both subtype A and B env proteins (P < 0.001; Table Table22).
Open in a separate windowaVariable-loop lengths and the numbers of PNGS in gp120 and gp41 within early HIV-1 env variants from subtypes A, B, C, and D characterized here and previously (2, 14, 15). n, number of samples.bKruskal-Wallis equality-of-populations rank test (based on multiple comparisons; P values of <0.007 were considered significant; significant values are presented in boldface).cWilcoxon rank sum test (based on multiple comparisons; P values of <0.008 were considered significant; significant values are presented in boldface).We then assessed the neutralization sensitivity of the pseudoviruses to antibodies in plasma from HIV-1-infected individuals and to HIV-1-specific MAbs by using the TZM-bl neutralization assay as described previously (2, 23, 38). Median inhibitory concentrations (IC50s) were defined as the reciprocal dilution of plasma or concentration of MAb that resulted in 50% inhibition of infection (2, 38). The Kenya pool was derived by pooling plasma collected between 1998 and 2000 from 30 HIV-1-infected individuals in Mombasa, Kenya, and the other three pools were derived by pooling plasma collected between 1993 and 1997 from 10 individuals from Nairobi, Kenya, and with an infection with a known subtype (A, C, or D) of HIV-1 as described previously (2).The env variants demonstrated a range of neutralization sensitivities to plasma samples, from neutralization resistant (defined as <50% neutralization with a 1:50 dilution of plasma) to neutralization sensitive with an IC50 of 333 (Fig. (Fig.2).2). Some clones, such as QF495.23M.ENV.A1, were relatively sensitive to all the plasma pools, with IC50s from 100 to 333, whereas other clones, such as QH343.21M.ENV.A10, were relatively resistant to these plasma pools, with IC50s from <50 to 85 (Fig. (Fig.2).2). The plasma pools did differ in their neutralization potencies. The Kenya pool, with a median IC50 of <50 across all viruses tested, was significantly less likely to neutralize these transmitted variants than were the subtype A, C, and D plasma pools, which had median IC50s of 110, 105, and 123, respectively (P values of <0.0001, 0.0001, and 0.001, respectively, by paired t test on log-transformed IC50s). The basis for these differences in neutralizing activity is not clear, although the location, timing, and level of immunodeficiency at the time of sample collection could have contributed to the differences in NAb levels between the pools.Open in a separate windowFIG. 2.Neutralization sensitivity of early subtype A, C, D, and A/D recombinant env variants to plasma samples and MAbs in relation to the sequences of the MAb binding sites. The env used to generate the pseudovirus tested is shown at the left, and the plasma pool or MAb tested is indicated at the top. The IC50s of each plasma sample or MAb against each viral pseudotype is shown, with darker shading indicating more potent neutralization, as defined at the bottom of the figure. Gray boxes indicate that <50% neutralization was observed at the highest dilution of plasma or concentration of MAb tested. Each IC50 shown is an average of the results from two independent neutralization assays, using pseudovirus generated in independent transfection experiments. The median IC50s from the 31 variants are shown at the bottom. Neutralization of the pseudovirus derived from the subtype B variant SF162 is shown as a control, and neutralizations of murine leukemia virus (MLV) and simian immunodeficiency virus clone 8 (SIV) are shown as negative controls. In the panels on the right, the sequences for the MAbs 2G12, 2F5, and 4E10 are displayed. For 2G12, the amino acid numbers for the five PNGS that are important for 2G12 binding are shown for each virus tested. A plus sign indicates that the PNGS at that site in the envelope sequence was preserved, and a minus sign indicates that the PNGS was deleted. A shift in the PNGS position is indicated by the amino acid position to which the PNGS shifted. All sequences were numbered relative to the HXB2 sequence. The two rightmost panels show data for the canonical 2F5 and 4E10 epitopes, with a period indicating that the amino acid is preserved.The env variants were significantly more susceptible to their subtype-matched plasma pool, with a higher mean IC50 for subtype-matched plasma samples than for unmatched plasma samples (138 versus 108, P = 0.0081, paired t test). However, a significant amount of cross-subtype neutralization was observed, as every env variant that was susceptible to the subtype-matched plasma pool was also susceptible to at least one of the other plasma pools (Fig. (Fig.2).2). Thus, although potency was enhanced when the plasma antibodies were produced in response to infection with the same subtype of HIV-1, there were shared neutralization determinants between subtypes, as has been observed previously (reviewed in reference 3).To identify potential correlates of neutralization sensitivity to the antibodies within these plasma pools, we included these 31 env variants and an additional 15 subtype A env variants we previously characterized from the same cohort with the same plasma pools (2). We did not observe a change in neutralization sensitivity during the evolution of the HIV-1 epidemic in Kenya, as no correlation was observed between neutralization sensitivity and the calendar date from which the env variants were isolated. In addition, no correlation was observed between the neutralization sensitivity of a variant to the plasma pools and the duration of estimated infection within that individual. Finally, there was no significant correlation between the neutralization sensitivity and variable-loop length or the number of PNGS. Thus, although changes in the variable-loop length or number of PNGS may alter the exposure of epitopes within the HIV-1 env protein, these changes do not appear to be the primary determinant of neutralization sensitivity.Despite relatively universal sensitivity to at least one of the pooled plasma samples, these transmitted Kenyan env variants were generally resistant to the MAbs 2G12 (provided by Hermann Katinger, Polymun Scientific) and b12 (provided by Dennis Burton, The Scripps Research Institute), as well as 2F5 and 4E10 (obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health) (Fig. (Fig.2),2), though these MAbs neutralized the subtype B env variant SF162, with IC50s similar to those reported previously (1). Subtype D strains were the most susceptible to MAbs, with 4/8 variants neutralized with <20 μg/ml of 2F5 and 2/8 neutralized with <20 μg/ml of the other MAbs. This could reflect the fact that subtype D variants are more closely related to subtype B strains (Fig. (Fig.1)1) (see reference 10), and these MAbs were all derived from subtype B-infected individuals.Among all 31 variants, 2F5 was the most broadly neutralizing, with 15/31 variants from 8/14 subjects neutralized with <20 μg/ml of this MAb. Some 2F5-resistant env variants, such as QH209.14M.ENV.A2 and QB857.110I.ENV.B3, had mutations in the canonical 2F5 binding epitopes, though other 2F5-resistant env variants such as QF495.23M.ENV.A3 and QA790.204I.ENV.A4 maintained the canonical 2F5 epitope. The results with the MAb 4E10 were similar; 4E10 neutralized only seven variants from 4 of the 14 subjects, and the presence of mutations in the 4E10 epitope, which were common, did not predict neutralization sensitivity (Fig. (Fig.2).2). For instance, the env variants QH343.21M.ENV.A10 and QH343.21M.ENV.B5 contained identical N671S and D674S mutations and QH343.21M.ENV.B5 was highly sensitive to 4E10, while QH343.21M.ENV.A10 was resistant (Fig. (Fig.2).2). Thus, for the 2F5 and 4E10 epitopes, the presumed epitopes appear to be shielded in a subset of these early non-subtype B env variants, as has been previously observed (Fig. (Fig.2)2) (1, 2, 5, 14).The MAb b12 neutralized only two variants from two subtype D-infected individuals, with no neutralization of the subtype A, C, and A/D recombinant pseudoviruses. Only four variants from two subjects were neutralized by 2G12 at <20 μg/ml, and these were the only variants that maintained all five of the PNGS within the 2G12 epitope (Fig. (Fig.2).2). Overall, the median IC50 of all the MAbs against these transmitted variants was >20 μg/ml. None of the variants was susceptible to all four MAbs (Fig. (Fig.2),2), unlike many of the early subtype B env variants characterized previously (14).In summary, these newly characterized HIV-1 env clones represent a range of neutralization sensitivities and can be used to supplement existing panels of transmitted variants, in particular, adding the first subtype D and A/D recombinant variants. Some differences between subtypes in env structure following transmission were noted, though these differences did not correlate with neutralization sensitivity. Although the significant levels of cross-subtype neutralization sensitivity observed with plasma samples indicate that some neutralization determinants were shared across subtypes, the epitopes for the MAbs b12, 2G12, 2F5, and 4E10 did not appear to be among the shared determinants. Thus, despite the fact that significant attention has focused on using vaccination to develop antibodies that resemble these MAbs in their specificity, such antibodies may not neutralize the transmitted strains that are causing most new infections worldwide. These data therefore stress the importance of evaluating transmitted variants in endemic areas when designing immunogens and evaluating vaccine and microbicide strategies. 相似文献
TABLE 1.
Demographic characteristics, diversities, gp120 variable-region lengths, numbers of PNGS, and accession numbers of cloned env variantsSubject | Virus subtype | Sample date (mo/day/yr) | dpia | Plasma VLb | Sourcec | Individual env clone | Pairwise difference (%)d | Variable-loop length (aa)
| No. of PNGS
| GenBank accession no. | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
V1/V2 | V3 | V4 | V5 | gp120 | gp41 | gp41 ecto | |||||||||
QB726 | A | 04/16/96 | 70 | 61,940 | ucPBMC | QB726.70M.ENV.B3 | 0.16 | 63 | 35 | 36 | 10 | 22 | 4 | 4 | FJ866111 |
QB726.70M.ENV.C4 | 63 | 35 | 36 | 10 | 22 | 4 | 4 | FJ866112 | |||||||
QF495 | A | 05/16/06 | 23 | 217,050 | ucPBMC | QF495.23M.ENV.A1 | 0.12 | 107 | 35 | 37 | 11 | 30 | 4 | 4 | FJ866113 |
QF495.23M.ENV.A3 | 107 | 35 | 37 | 11 | 30 | 4 | 4 | FJ866114 | |||||||
QF495.23M.ENV.B2 | 113 | 35 | 37 | 11 | 31 | 4 | 4 | FJ866115 | |||||||
QF495.23M.ENV.D1 | 113 | 35 | 37 | 11 | 31 | 4 | 4 | FJ866116 | |||||||
QG984 | A | 07/12/04 | 21 | 30,300 | ucPBMC | QG984.21M.ENV.A3 | NA | 69 | 34 | 36 | 11 | 24 | 3 | 3 | FJ866117 |
QH209 | A | 10/13/05 | 14 | 28,600 | ucPBMC | QH209.14M.ENV.A2 | NA | 72 | 35 | 29 | 11 | 24 | 4 | 4 | FJ866118 |
QH343 | A | 09/08/05 | 21 | 40,750,000 | ucPBMC | QH343.21M.ENV.A10 | 0.19 | 77 | 35 | 32 | 15 | 26 | 4 | 4 | FJ866119 |
QH343.21M.ENV.B5 | 77 | 35 | 32 | 15 | 26 | 4 | 4 | FJ866120 | |||||||
QH359 | A | 10/05/05 | 21 | 32,120 | ucPBMC | QH359.21M.ENV.C1 | 1.4 | 84 | 35 | 36 | 10 | 29 | 4 | 4 | FJ866121 |
QH359.21M.ENV.D1 | 73 | 35 | 35 | 10 | 26 | 4 | 4 | FJ866122 | |||||||
QH359.21M.ENV.E2 | 72 | 35 | 40 | 13 | 28 | 4 | 4 | FJ866123 | |||||||
QA790e | A/D | 06/10/96 | 204 | 48,100 | ccPBMC | QA790.204I.ENV.A4 | 0.36 | 77 | 35 | 33 | 11 | 25 | 4 | 4 | FJ866124 |
QA790.204I.ENV.C1 | 77 | 35 | 33 | 11 | 26 | 4 | 4 | FJ866125 | |||||||
QA790.204I.ENV.C8 | 77 | 35 | 33 | 11 | 24 | 4 | 4 | FJ866126 | |||||||
QA790.204I.ENV.E2 | 77 | 35 | 33 | 11 | 25 | 4 | 4 | FJ866127 | |||||||
QG393 | A2/D | 06/23/04 | 60 | 17,360 | ucPBMC | QG393.60M.ENV.A1 | 0.7 | 60 | 34 | 31 | 10 | 24 | 5 | 5 | FJ866128 |
QG393.60M.ENV.B7 | 57 | 34 | 31 | 10 | 24 | 5 | 5 | FJ866129 | |||||||
QG393.60M.ENV.B8 | 57 | 34 | 31 | 10 | 24 | 5 | 5 | FJ866130 | |||||||
QB099e | C | 02/10/95 | 391 | 27,280 | ucPBMC | QB099.391M.ENV.B1 | 0.43 | 65 | 35 | 29 | 10 | 25 | 4 | 4 | FJ866131 |
QB099.391M.ENV.C8 | 65 | 35 | 29 | 10 | 25 | 4 | 4 | FJ866132 | |||||||
QC406 | C | 07/08/97 | 70 | 692,320 | ucPBMC | QC406.70M.ENV.F3 | NA | 64 | 35 | 20 | 11 | 22 | 5 | 4 | FJ866133 |
QA013 | D | 10/11/95 | 70 | 1,527,700 | ccPBMC | QA013.70I.ENV.H1 | 0.16 | 60 | 34 | 29 | 12 | 25 | 4 | 4 | FJ866134 |
QA013.70I.ENV.M12 | 60 | 34 | 29 | 12 | 25 | 4 | 4 | FJ866135 | |||||||
QA465 | D | 08/19/93 | 59 | 37,750 | ucPBMC | QA465.59M.ENV.A1 | 0.24 | 65 | 35 | 30 | 11 | 28 | 4 | 4 | FJ866136 |
QA465.59M.ENV.D1 | 65 | 35 | 30 | 11 | 27 | 4 | 4 | FJ866137 | |||||||
QB857 | D | 10/16/97 | 110 | 14,640 | ucPBMC | QB857.23I.ENV.B3 | NA | 68 | 34 | 32 | 11 | 26 | 5 | 4 | FJ866138 |
QD435 | D | 04/06/99 | 100 | 17,470 | ucPBMC | QD435.100M.ENV.A4 | 0.88 | 69 | 34 | 29 | 12 | 26 | 5 | 4 | FJ866139 |
QD435.100M.ENV.B5 | 67 | 34 | 29 | 11 | 24 | 5 | 4 | FJ866140 | |||||||
QD435.100M.ENV.E1 | 69 | 34 | 29 | 12 | 26 | 5 | 4 | FJ866141 |
TABLE 2.
Summary of variable-loop lengths and the numbers of PNGS in gp120 and gp41 within early HIV-1 env variantsaParameter | Median value (25th percentile, 75th percentile) for subtype:
| Kruskal- Wallis P valueb | Wilcoxon rank sum P values for individual comparisonsc
| ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
A (n = 11) | B (n = 19) | C (n = 20) | D (n = 4) | A vs. B | A vs. C | A vs. D | B vs. C | B vs. D | C vs. D | ||
Length | |||||||||||
V1/V2 | 70.3 (62, 76) | 70 (66, 70) | 65 (62, 76) | 66.5 (62, 69) | 0.21 | 0.730 | 0.282 | 0.215 | 0.051 | 0.113 | 0.846 |
V3 | 35 (34, 35) | 35 (35, 35) | 35 (34, 35) | 34 (34, 35) | 0.001 | 0.240 | 0.016 | 0.107 | 0.141 | 0.006 | <0.001 |
V4 | 32 (30, 36) | 33 (31, 34) | 26.5 (22, 29) | 29.5 (29, 31) | 0.0001 | 0.880 | <0.001 | 0.148 | <0.001 | 0.023 | 0.056 |
V5 | 11 (11, 11) | 10 (9, 11) | 10 (9, 11) | 11.5 (11, 12) | 0.030 | 0.096 | 0.015 | 0.184 | 0.677 | 0.099 | 0.021 |
No. of PNGS in: | |||||||||||
gp120 | 24 (23, 28) | 25 (24, 26) | 24 (23, 25) | 26 (26, 27) | 0.20 | 0.680 | 0.692 | 0.265 | 0.146 | 0.186 | 0.042 |
gp41 | 4 (4, 5) | 5 (4, 5) | 5 (4, 5) | 4.5 (4, 5) | 0.20 | 0.030 | 0.179 | 0.470 | 0.410 | 0.408 | 0.799 |
gp41ecto | 4 (4, 4) | 4 (4, 4) | 4 (4, 5) | 4 (4, 4) | 0.044 | 0.107 | 0.025 | 0.550 | 0.088 | 0.507 | 0.201 |
10.
We present 2 cases of Niemann Pick disease, type B with secondary sea-blue histiocytosis. Strikingly, in both cases the Pick cells were positive for tartrate resistant acid phosphatase, a finding hitherto described only in Gaucher cells. This report highlights the importance of this finding as a potential cytochemical diagnostic pitfall in the diagnosis of Niemann Pick disease.Key words: Niemann pick disease, Gaucher disease, tartrate resistant acid phosphatase, sea blue histiocytosis.We present two unrelated patients who were referred to the Hematology OPD from Gastroenterology during work-up of long-standing splenomegaly 2 years apart and whose details are presented in Patient 1 Patient 2 Age, sex 14 yr/F 18 yr/F Presenting complaints Pain, awareness of mass in left upper abdomen ×12 years Low grade fever on and off, abdominal discom fort ×2 yrs Hb (gm%), TLC (/µL), platelets (/µL) 7.3, 4500, 153000 12, 6900, 47000 Liver / Spleen Not palpable / 14 cm below costal margin Not palpable / massive enlargement (span 20 cm) Ultrasound abdomen Massive splenomegaly, multiple hyperechoeic foci, no evidence of EHPVO or HVOTO Splenomegaly, mesenteric lymphadenopathy CECT abdomen Not done Splenomegaly, pre-aortic lymphadenopathy (? lymphoma infiltration) total protein, Albumin, urea, creatinine, sodium, potassium Serum bilirubin, alkaline phosphatase, SGOT, SGPT, Normal ranges Normal ranges Hemoglobin HPLC, direct and indirect antiglobulin tests, 24-hour incubated osmotic fragility test, G6PD deficiency screening Normal Normal RK-39 antigen test for Leishmaniasis, HBsAg, anti HCV, anti HIV 1 & 2 Negative Negative HDL Cholesterol (normal 40–50 mg%) 12 mg% 23 mg% Fundoscopic examination Normal Bilateral cherry red spots Acid phosphatase (normal >6.5 U/L) 5.5 U/L 4.2 U/L Bone marrow examination Aspirate: Cellular smears with normal marrow Diluted marrow with many foamy elements, foamy histiocytes present along with numerous sea blue histiocytes, some foamy histiocytes show haemophagocytosis Biopsy: hypercellular, foamy cells and other histiocytes prominent histiocytes and sea blue histiocytes, normal marrow elements seen Biopsy: normocellular, foamy cells and other histiocytes present Cytochemistry Foamy cells positive for Sudan Black B, acid phosphatase (AP), tartrate resistant acid phosphatase (TRAP), weak hue with periodic acid Schiff (PAS), sea blue histiocytes strongly positive for PAS and AP Foamy cells positive for Sudan Black B, acid Phosphatase, TRAP, weak positive with PAS; sea blue histiocytes positive for PAS, AP Enzyme assay Normal beta-glucocerebrosidase level, sphingomyelinase- not done Normal beta-glucocerebrosidase level, sphingomyelinase- 9 nmol/17 hr/mg protein (normal 10–47 nmol/17 hr/mg protein)