CCR5 and CXCR4 usage by non-clade B human immunodeficiency virus type 1 primary isolates

CCR5 and CXCR4 usage has been studied extensively with a variety of clade B human immunodeficiency virus type 1 (HIV-1) isolates. The determinants of CCR5 coreceptor function are remarkably consistent, with a region critical for fusion and entry located in the CCR5 amino-terminal domain (Nt). In particular, negatively charged amino acids and sulfated tyrosines in the Nt are essential for gp120 binding to CCR5. The same types of residues are important for CXCR4-mediated viral fusion and entry, but they are dispersed throughout the extracellular domains of CXCR4, and their usage is isolate dependent. Here, we report on the determinants of CCR5 and CXCR4 coreceptor function for a panel of non-clade B isolates that are responsible for the majority of new HIV-1 infections worldwide. Consistent with clade B isolates, CXCR4 usage remains isolate dependent and is determined by the overall content of negatively charged and tyrosine residues. Residues in the Nt of CCR5 that are important for fusion and entry of clade B isolates are also important for the entry of all non-clade B HIV-1 isolates that we tested. Surprisingly, we found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects fusion and entry of all non-clade B isolates tested. This points to a different mechanism of CCR5 usage by these viruses and may have important implications for the development of HIV-1 inhibitors that target CCR5 coreceptor function.     

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1–4 CELLS

Abstract  The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B1–4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins, however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B1–4 cells. The growth characteristics, productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B1–4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing ß -galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B1–4 cells.     

RMND5B基因的克隆及其腺病毒载体的构建

已有研究表明RMND5B可能与心肌肥大相关,但具体机制不明,RMND5B的重组腺病毒载体的构建和鉴定为研究RMND5B功能提供了基础工具。首先,设计小鼠RMND5B基因的特异性引物,以c DNA为模板,PCR扩增RMND5B ORF区,并在两端各加入HindⅢ及SalⅠ的酶切位点。将此片段插入到p MD18-T载体,再亚克隆至线性化的穿梭质粒p Ad Track-CMV中。PmeⅠ酶切线性化之后,电转化到含p Ad Easy-1的BJ5183感受态细菌中。在BJ5183细菌中发生同源重组获得了重组质粒p Ad-RMND5B质粒。PacⅠ酶切线性化之后转染293A细胞,经过包装获得腺病毒AdRMND5B。将此腺病毒感染新生大鼠原代心肌细胞,并在一段时间后观察绿色荧光并通过RT-PCR检测RMND5B的表达情况。结果表明,成功构建了RMND5B的重组腺病毒载体并实现了AdRMND5B在新生大鼠原代心肌细胞中表达,为进一步研究RMND5B基因在心肌肥大中的作用奠定了良好的实验基础。     

Effects of 5-fluorouracil on B lymphocyte lineage cells

The aim of these studies was to determine the sensitivity of B lymphocyte lineage precursor cells in mice to 5-fluorouracil (5-FU). Selective effects could be very helpful in dissecting precursor-product relationships between these and the rare multipotential stem cells from which they derive. Numbers and functions of particular types of cells were determined at intervals after a single treatment (3 mg) and, as expected, myeloid-committed stem cells were very severely affected. Day 8 spleen colony-forming cells (CFU-S) and colony-stimulating factor-responsive macrophage progenitors were reduced by 98% within 24 hr, whereas presumptive early stem cells (day 14 CFU-S) were much more resistant. B cells, which were probably recently formed in bone marrow and which are not thought to be actively dividing, were also 5-FU-sensitive, but perhaps less so than pre-B cells and immunoglobulin-negative lymphocytes bearing a B lineage marker. Approximately 5 wk were required for the normal cellular composition of marrow to return to normal. Transplantation of marrow from 5-FU-treated mice suggested that the slow regeneration of B lymphocytes might partially result from residual drug effects or damage to microenvironmental elements which are required for B lineage differentiation. Acute reductions of lymphocytes in the thymus were also documented, and the larger cells declined most rapidly and regenerated most slowly in that tissue. Of particular interest was the differential susceptibility of B cells in various lymphoid tissues to 5-FU. Whereas lymph node B cells were minimally affected, one-half of the splenic B cells disappeared within 48 hr of drug injection. Intrinsic differences in 5-FU sensitivity were confirmed by treatment of lymphocytes in vitro, and this suggests that particular B cell sets may be metabolically distinct. This drug should find additional experimental application in studies of B lymphocyte formation and functional heterogeneity.     

Human B lymphocytes possess 5-lipoxygenase activity and convert arachidonic acid to leukotriene B4.

Incubation of cell sonicates from monoclonal B cells with arachidonic acid led to the formation of leukotriene (LT) B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In contrast, stimulation of intact B cells with the calcium ionophore A23187 +/- arachidonic acid did not, under similar conditions, lead to formation of LTB4. The identification of these products was based on reverse phase- and straight phase-HPLC analysis, UV-spectroscopy and gas chromatography-mass spectrometry. Cell sonicates of highly enriched human tonsillar B lymphocytes also converted arachidonic acid to LTB4 and 5-HETE. Activation of these cells with B cell mitogen and cytokines for three days led to an upregulation of 5-lipoxygenase activity. This study provides evidence for the biosynthesis of LTB4 from arachidonic acid in B cell lines and in normal human tonsillar B lymphocytes.     

Structure and dimerization of translation initiation factor aIF5B in solution

Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s(20,w)(0) were determined to be 3.64 and 5.51±0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5±0.2 ? and a maximum dimension of ~130 ?. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli.     

5-Hydroxy-2-(2-phenylethyl)chromone (5-HPEC): A novel non-nitrogenous ligand for 5-HT2B receptor

Chromones are a class of natural products found in almost every known terrestrial plant with over 4000 naturally occurring derivatives having been isolated and structurally elucidated. Recently, 5-hydroxy-2-(2-phenylethyl)chromone (5-HPEC), isolated from Imperata cylindrical, showed neuroprotective activity against glutamate induced excitotoxicity in primary cultures of rat cortical cells. In comparison to other naturally occurring neuroprotective chromones, 5-HPEC contains fewer hydroxyl groups. Here we report our most recent characterization on this interesting natural product against a number of CNS receptors for the purpose to identify the potential molecular targets that may be related to its biological activity. Based on our studies, including radiobinding assays, calcium flux functional assays and molecular modeling studies, 5-HPEC may represent a type of novel nonnitrogenous ligands to the 5-HT2B receptor.     

Estrogen induces normal murine CD5+ B cells to produce autoantibodies

Females have better humoral immune responses and are more susceptible to autoimmune diseases than males. Normal female mice (C57BL/6J, C3H/HeJ, and NZW) have significantly increased spontaneous autoimmune plaque-forming cells (APFC) to mouse erythrocytes pretreated with bromelain (Br-ME) in spleen, peritoneal exudate cell, and bone marrow compared to their male counterparts. A minor subpopulation of B cells, CD5+ B, is thought to produce this autoantibody. As determined by dual color flow cytometry, increased APFC to Br-ME in females is not due to quantitative increase of CD5+ B cells. Rather, it is due to increased numbers or percentages) of CD5+ B cells producing these autoantibodies, because CD5+ B cells from females produced greater numbers of APFC to Br-ME than equal numbers of cells derived from males. The increased autoantibody production in females can be attributed to the effect of estrogen on the immune response because this hormone markedly augments APFC to Br-ME in intact or orchidectomized males. Male hormone had little effect. Importantly, estrogen did not increase the numbers of B or CD5+ B cells but augmented the ability of B cells to produce this response. This was verified when a T cell-depleted B cell fraction or fluorescence-activated cell sorter purified CD5+ B cells from estrogen-treated mice proved more efficient in the production of APFC to Br-ME. These results suggest that the number of CD5+ B cells committed to produce autoantibodies to Br-ME is increased under the influence of estrogen. This is the first demonstration that estrogen can augment the production of natural autoantibodies in normal mice. The overall augmented humoral immune responses in females and the B cell hyperactivity in female predominant autoimmune diseases appears to be due to estrogen.     

Distribution of IL-5 receptor-positive B cells. Expression of IL-5 receptor on Ly-1(CD5)+ B cells.

mAb to murine IL-5R were prepared by means of fusion between mouse myeloma cells and spleen cells from a rat immunized with membrane-enriched fractions of IL-5-dependent early B cell line (T88-M). Two mAb (H7 and T21) were selected for their competitive inhibition of receptor binding by 35S-labeled IL-5 and of IL-5 biologic activities. The number of binding sites recognized by the mAb on different cell lines correlated with IL-5 responsiveness. Most surface IgM+ peritoneal B cells were H7+ and more than 70% were also Ly-1(CD5)dull+, and responded to IL-5 for polyclonal IgM production in a high frequency. A significant proportion of splenic B cells reacted with these mAb, although lower number (one-log less) than peritoneal B cells and a small proportion of H7dull+ splenic B cells seems to be Ly-1(CD5)dull+, 1 of 200 splenic B cells responded to IL-5 for IgM production. These results suggest that IL-5R+ B cells may consist of a subpopulation of B cells. Intriguingly, lymphoid populations of bone marrow cells were stained with H7 and T21, whereas myeloid populations were brightly stained with only T21. Finally, both H7 and T21 mAb specifically precipitated a protein of a Mr 60,000 from 125I-labeled cell lysates of IL-5R+ T88-M cells. The IL-5R with similar size (Mr 55,000 to 60,000) was precipitated from the cell lysates of peritoneal B cells. T21 mAb but not H7 mAb precipitated a protein of a Mr 110,000 from the cell lysates of bone marrow cells.     

Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix

Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea‐specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes. Proteins 2016; 84:712–717. © 2016 Wiley Periodicals, Inc.     

Preparation and mass spectral behaviour of some 5 beta-cholenoic acids.

Monounsaturated 5 beta-cholanoic acids with double bonds in rings A, B, and C were prepared by POCl3 and ZnCl2 dehydration from natural bile acids with selectively blocked hydroxyl functions. The yields ranged from 15 to 100%. The products were purified by thin-layer and AgNO3 thin-layer chromatography and the structures were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. The methyl ester acetates of the unsaturated 5 beta-cholanoic acids possessed chromatographic properties closely similar to those of the corresponding saturated bile acids. Several characteristic fragments were seen in the mass spectra which, in conjunction with the chromatographic properties, permitted an unambiguous distinction between different monounsaturated acids, and between saturated and unsaturated bile acids of the same number and configuration of functional groups. The 20 5 beta-cholenoic acids examined represent all of the simple chemical and enzymatic dehydration products of natural bile acids and can be completely identified by their combined chromatographic and mass spectral properties.     

Structure and activity of insulin, XIII. Specificity of the arginine-guanidino group in biologically active tetrapeptides of the insulin sequence B 22-25 (Arg-Gly-Phe-Phe).

The biologically active partial sequence Arg-Gly-Phe-Phe (position B 22-25 of the insulin B chain) in the form of the synthetic tetrapeptidamide, was compared in several bioassays with the following analogous synthetic peptides: homoarginyl-, ornithyl-, lysyl-, citrullyl-, alanyl- and NG-nitroarginyl-Gly-Phe-Phe-NH2. The syntheses of the lysyl- and alanyl-tetrapeptidamides are described. After intraperitoneal injection of the peptides in doses of 3-100 mumol per 100 g rat, together with [U-14C]glucose, the natural sequence Arg-Gly-Phe-Phe showed the highest insulin like activity (incorporation of labeled carbon into the diaphragm). The activity of the homoarginyl peptide was a little weaker. The ornithyl- and the lysyl-peptide, however, showed a remarkably diminished activity. The activity of the citrullyl-peptide was even lower and the alanyl-peptide was inactive. In vitro assays with rat diaphragm showed the same range of effects for the elevation of glucose uptake and glycogen content of the diaphragm. The activity decreased in the following order: Arg- greater than Har- greater than Orn- greater than Cit-Gly-Phe-Phe-NH2. Alanyl- and Nitroarginyl-Gly-Phe-Phe-NH2 were without effect. In isolated fat cells the glucose oxidation was enhanced significantly only by the arginyl-peptide. The results show that among the structures examined the guanidino group carried by the C5 chain of arginine is the most effective. The results are in accordance with our preceding work [1] using semisynthetic insulins obtained from natural A-chain and synthetic B-chain variants. In these products the replacement of Arg B 22 by ornithine or lysine also led to drastically diminished activity and after replacement of Arg B 22 by alanine the activity also disappeared.     

Genetic influence on the levels of circulating CD5 B lymphocytes

By using sensitive two- and three-color immunofluorescence analyses, we readily detect CD5B cells (Leu 1 B cells) in the peripheral blood of normal adults. These circulating CD5 B lymphocytes coexpress B cell differentiation antigens CD20, CD21, CD19, sIgM and sIgD, and HLA-DR. Unlike CD5-negative B cells from most adults, however, these cells co-express CD11, a finding also noted for malignant CD5 B cells from several patients with CLL. Between normal volunteers, there exists heterogeneity in the proportion of PBL that co-express CD5 and B cell surface antigens, such cells representing between 0 and 6% of peripheral lymphocytes. Despite such heterogeneity between unrelated individuals, analyses of repeated blood samples from the same person reveal that the proportions of CD5 B lymphocytes are constant over time. Examination of blood samples from related family members, monozygotic twins, and triplets indicate that the relative proportion of circulating CD5 B cells may be genetically regulated. This is apparent even for monozygotic twins discordant for rheumatoid arthritis. Four sets of such twins are examined, each set having one individual with clinically active, seropositive rheumatoid arthritis and another without detectable rheumatoid factor or clinical pathology. Despite such noted differences, twins from each set share identical proportions of circulating CD5 B cells. In summary, our studies indicate that the level of CD5 B lymphocytes is a rather stable phenotypic trait that is under genetic control.     

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C)

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

B5, a new B cell-restricted activation antigen

The characterization of a new human B cell-restricted activation antigen (B5) is described in this report. With the use of a monoclonal antibody to B5, we show that B5 can be detected on peripheral blood or splenic B cells after 1 day of stimulation with either anti-immunoglobulin, protein A, Epstein Barr virus, or pokeweed mitogen. In contrast, B5 was not expressed on resting B, T, or myeloid cells. More important, B5 could not be detected on activated T cells or monocytes. The B5 antigen was expressed on some lymphoblastoid B cell lines and B cell neoplasms but was not expressed on leukemias or lymphomas of T or myeloid origin. The B5 antigen is distinct from previously reported B cell activation antigens by its m.w. and pattern of cellular expression. These studies suggest that B5 is a novel B cell-restricted activation antigen, which may be useful to study the events of early human B cell activation.     

5-oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.     

Responses of B cells from autoimmune mice to IL-5

Three strains of mice (NZB/W F1 X NZW (NZB/W), BXSB, and MRL/Mp-lpr/lpr (MRL/lpr] develop an autoimmune disease that is clinically and immunologically similar to human SLE. A characteristic of these mice is polyclonal B cell hyperactivity. To explore whether this may be related to hyper-responsiveness to B cell stimulatory factors, we investigated the proliferative and secretory responses of B cells from these mice to semi-purified natural and rIL-5, a major regulator of B cell development in the mouse. As this lymphokine stimulates growth and differentiation of activated B cells, attention was focused on in vivo-activated B cell populations, obtained from the interface of 50/65% Percoll density gradients, from normal or autoimmune mice. This cell population from NZB/W mice secreted IgM and incorporated [3H]TdR at significantly higher levels in response to IL-5, and was more sensitive to IL-5, than a comparable population from several normal murine strains. NZB/W female and male mice displayed heightened responses to IL-5, indicating that this is characteristic of the strain in general and is not associated with the accelerated severe disease of the females. Small resting B cells from NZB/W and normal mice were insensitive to IL-5 stimulation. In contrast to NZB/W mice, no difference was observed in the magnitude of either proliferative or Ig secretory responses between in vivo-activated B cell populations from autoimmune BXSB and MRL/lpr or normal mice. Thus, B cell hyper-responsiveness to IL-5 is a characteristic of NZB/W mice but not of two other lupus-prone murine strains. As one unique feature of NZB/W mouse B cells compared to normal and other autoimmune B cells is an elevated proportion of Ly-1+ B cells, the possibility of IL-5 hyper-responsiveness being associated with this B cell subpopulation was investigated. Fluorescence-activated cell sorter sorted Ly-1+ and Ly-1- B cells both responded to IL-5, however Ly-1+ B cells consistently showed a higher stimulation index in both proliferative and Ig secretory responses to this lymphokine.     

STAT5 activation underlies IL7 receptor-dependent B cell development

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R(-/-) background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R(-/-) mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-x(L) expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R(-/-) mice as determined by 1) the restoration of V(H) Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.