Production, characterization and antioxidant activity of exopolysaccharides from submerged culture of Morchella crassipes

The aim of this work was to investigate the fermentation optimization, molecular characterization, and antioxidant activity in vitro of exopolysaccharides (EPS) from Morchella crassipes in submerged culture. Firstly, an optimal medium for EPS production was obtained by single-factor experiment and central composite design as follows: maltose 44.79?g/L and tryptone 4.21?g/L. Then, one fraction of EPS was obtained from the culture filtrates by size exclusion chromatography and the molecular characteristics were examined by a multi-angle laser light scattering and refractive index detector system. The weight-average molar mass and the polydispersity ratio of the EPS fraction were revealed to be 1.961?×?10(4)?g/mol and 1.838, respectively. FT-IR spectroscopy was used for obtaining vibrational spectra of the purified EPS fraction. Finally, the antioxidant activity of EPS was investigated and the relationship with molecular properties was discussed as well.     

Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals.

The rate of clinical progression is variable among individuals infected with human immunodeficiency virus type 1 (HIV-1). Changes in viral burden which correlate with disease status have been demonstrated in cross-sectional studies; however, a detailed longitudinal study of the temporal relationship between viral burden, CD4+ T-cell numbers, and clinical status throughout the course of infection has not been reported. Multiple longitudinal blood samples were obtained from four HIV-1-infected individuals with clinically divergent profiles. Levels of HIV-1 were measured in sequential samples of peripheral blood mononuclear cells, using both end-point dilution cultures and quantitative polymerase chain reaction methods. Serial HIV-1 isolates from each case were also evaluated to determine their biological properties in vitro. For the three patients with clinical progression, a dramatic increase in the level of HIV-1 was observed concurrent with or prior to a marked drop in CD4+ T lymphocytes. This increase in viral burden was temporally associated with the emergence of a more cytopathic viral phenotype. In contrast, consistently low levels of HIV-1 were observed in the one patient who was clinically and immunologically stable for more than a decade. Moreover, viral isolates from this patient were less cytopathic in vitro compared with HIV-1 isolates from those patients with disease progression. The temporal association between increased viral burden and CD4+ T-cell decline suggests a direct role for HIV-1 in the cytopathology of CD4+ T cells in vivo. Our results indicate that the pathogenic mechanisms responsible for CD4+ T-cell depletion may be related to both quantitative and qualitative changes in HIV-1.     

Discovery of the dual polysaccharide composition of emulsan and the isolation of the emulsion stabilizing component

Emulsan has been reported as an emulsion stabilizing amphipathic lipoheteropolysaccharide secreted by the oil-degrading bacterium Acinetobacter venetianus RAG-1. Previously, emulsan was regarded as a single polymer. As a result of developing a new purification process, we have discovered that emulsan is a complex of approximately 80% (w/w) lipopolysaccharide (LPS) and 20% (w/w) high molecular weight exopolysaccharide (EPS). The EPS was purified to 98% (w/w) using tangential flow filtration, Triton X-114 phase extraction, ammonium sulfate precipitation, and hydrophobic interaction chromatography. Several previously reported physical properties of emulsan can be attributed to the LPS fraction, such as charge, fatty acid profile, and solution behavior, while the EPS is responsible for the emulsion stabilization activity. The EPS is believed to be cationic in nature, thus providing an electrostatic binding mechanism for the formation of the emulsan complex.     

一株芽孢杆菌胞外多糖的分离纯化及其抗氧化性测定

基于实验室从新疆罗布泊沙漠筛选到一株芽孢杆菌, 研究了该菌胞外多糖的分离纯化工艺及其抗氧化性质。发酵液经离心, 抽滤等预处理后, 使用Sevag试剂除蛋白, 并以无水乙醇作提取溶剂, 通过正交实验确定最佳提取条件为: pH为7.0, 温度为4°C, 时间为1.5 h, 料液比为1:4。粗多糖溶解后上活性炭柱(1.5 cm ′ 24 cm), 用蒸馏水、60%乙醇及95%乙醇洗脱, 分离得到主要部分, 再经Sephadex G-100凝胶柱, 用0.2 mol/L的NaCl溶液洗脱, 硫酸苯酚法和考马斯亮蓝     

Rapid-high, syncytium-inducing isolates of human immunodeficiency virus type 1 induce cytopathicity in the human thymus of the SCID-hu mouse.

Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) disease is associated with an increased viral burden in the peripheral blood and a loss of circulating CD4+ T cells. HIV-1 isolates obtained prior to this stage of disease often have a "slow-low," non-syncytium-inducing (NSI) phenotype, whereas those obtained afterwards are often characterized as "rapid-high" and syncytium inducing (SI). Paired NSI and SI isolates from two different patients were inoculated into the human thymus implants of SCID-hu mice. The two slow-low, NSI isolates replicated to minimal levels in the grafts and did not induce thymocyte depletion. In contrast, the two SI isolates from the same patients showed high levels of viral replication and induced a marked degree of thymocyte depletion, accompanied by evidence of programmed cell death. These observations reveal a correlation between the replicative and cytopathic patterns of HIV-1 isolates in vitro and in the SCID-hu mouse in vivo and provide direct evidence that the biological phenotype of HIV-1 switch may be a causal and not a derivative correlate of HIV-1 disease progression.     

Analysis of the replication kinetics in primary culture of isolates of HIV-1 from infected patients in various stages of the disease]

The authors have studied the replicative kinetics and the induction of cytopathic effects of HIV-1 in primary co-cultures from infected subjects at various stages of the disease. Cultures from subjects with ARC or AIDS yielded HIV-1 replication more precociously and at higher levels compared to those from asymptomatic subjects; cytopathic effect "in vitro" were observed more frequently and earlier in cell cultures from ARC/AIDS subjects. Presented data indicate that the clinical and immunological deterioration during HIV-1 infection is related to viral replicative activity and suggest that the study of HIV-1 replicative kinetics in primary co-cultures may be helpful in predicting who will progress to AIDS.     

CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue

A rapid decline in T-cell counts and the progression to AIDS is often associated with a switch from CCR5-tropic (R5) HIV-1 to CXCR4-tropic (X4) HIV-1 or R5/X4 HIV-1 variants. Experimental infection with R5 HIV-1 causes less T-cell depletion than infection with X4 or R5/X4 variants in T-cell cultures, in ex vivo infected human lymphoid tissue and in SCID/hu mice, despite similar replication levels. Experimental genetic changes in those sequences in gp120 that transform R5 HIV-1 variants into otherwise isogenic X4 viruses make them highly cytopathic. Thus, it is now believed that R5 variants are less cytopathic for T cells than are X4 variants. However, it is not known why CCR5-mediated HIV-1 infection does not lead to a massive CD4+ T-cell depletion, as occurs in CXCR4-mediated HIV-1 infection. Here we demonstrate that R5 HIV-1 isolates are indeed highly cytopathic, but only for CCR5+/CD4+ T cells. Because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T-cell count. These results may explain why the clinical stage of HIV disease correlates with viral tropism.     

Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef.

nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process.     

A beta-galactose-specific lectin isolated from the marine worm Chaetopterus variopedatus possesses anti-HIV-1 activity

A 30 kDa beta-galactose-specific lectin named CVL was isolated from the polychaete marine worm Chaetopterus variopedatus (Annelida) and its anti-HIV-1 activity in vitro was determined. Results showed that CVL inhibited cytopathic effect induced by HIV-1 and the production of viral p24 antigen. The EC(50) values were 0.0043 and 0.057 microM, respectively. Time-of-addition analysis of anti-HIV-1 activity indicated its action was at the early stage of virus replication. CVL could blocked the cell-to-cell fusion process of HIV infected and uninfected cells with an EC(50) of 0.073 microM. The inhibition of HIV-1 entry into host cells was demonstrated by using fluorescence-based real-time quantify PCR. At CVL concentration of 0.33 microM and 0.07 microM, 86% and 21% virus attachment were blocked, respectively. The anti-HIV-1 action of CVL might relate to blockade of HIV-1 entry into cells.     

Human immunodeficiency virus infection of T-lymphoblastoid cells reduces intracellular pH.

Alterations in plasma membrane function are induced by many cytopathic viruses, including human immunodeficiency virus type 1 (HIV-1). These alterations can result in changes in the intracellular content of ions and other small molecules and can contribute to cytolysis and death of the infected cell. The pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethyl ester was used to quantitate intracellular pH (pHi) in HIV-1-infected T cells. Infection of cells from the CD4+ T-lymphoblastoid line HUT-78 (RH9 subclone) with HIV-1 strain LAI resulted in a significant decrease of pHi, from approximately 7.2 in mock-infected cells to below 6.7 by day 4 after infection, when cells were undergoing acute cytopathic effects. The pHi in persistently infected cells that survived the acute cytopathic effects of HIV-1 was approximately 6.8 to 7.0. Studies with amiloride, an inhibitor of the Na+/H+ exchange system, suggest that HIV-1-induced intracellular acidification in lymphocytes is due, in part, to dysfunction of this plasma membrane ion transport system. The alterations in pHi may mediate certain cytopathic effects of HIV-1, thereby contributing to depletion of CD4+ T lymphocytes in patients with AIDS.     

Cytoplasmic domain truncation enhances fusion activity by the exterior glycoprotein complex of human immunodeficiency virus type 2 in selected cell types.

To investigate the glycoprotein determinants of viral cytopathology, we constructed chimeric env genes between a noncytopathic strain of human immunodeficiency virus type 2 (HIV-2), designated HIV-2/ST, and a highly fusogenic and cytopathic variant derived from this virus. Expression of the resulting chimeric glycoproteins indicated that efficient syncytium formation in the human T-cell line Sup T1 mapped to the C-terminal region of the transmembrane (TM) glycoprotein subunit. In this region, the wild-type and cytopathic ST glycoproteins differed by only four amino acids and by the presence of a premature termination codon in the cytopathic variant. Subsequent site-directed mutagenesis indicated that the cytoplasmic domain truncation was responsible for the enhanced fusion activity. This modification, however, increased the fusion activity of the glycoprotein only in Sup T1 cells (in which the ST variant arose) but not in Molt 4 clone 8 or peripheral blood mononuclear cells. These observations indicate that the length of the cytoplasmic domain of the HIV-2 glycoprotein modulates the fusion activity of the exterior glycoprotein complex in a cell-specific manner. Such adaptability appears to permit the emergence of fusogenic variants during HIV-2 passage in vitro and may also regulate viral growth or cytopathic effects in selected cell types during natural infection in vivo.     

Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI.

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.     

一株红树林真菌抗单纯疱疹病毒Ⅱ型的研究

目的:对分离自海南红树林真菌菌株PH0016的胞外多糖(exopolysaccharide,EPS)抗单纯疱疹病毒Ⅱ型(Herpes simplex virus type 2,HSV-2)活性进行研究。方法:收集纯化菌株PH0016产生的EPS,体外采用细胞病变观察EPS的细胞毒性和抗HSV-2作用。为观察EPS对HSV-2体内感染的治疗效果,小鼠颅内注射0.02ml滴度为100LD50/0.1ml-1的HSV-2,24小时后以不同浓度EPS灌胃,持续7天,14天后计算小鼠存活率和存活时间。菌株分类采用形态学观察和ITS序列分析。结果:EPS可抑制HSV-2病毒活性,病毒感染的细胞病变的半抑制浓度(Inhibited concentration,IC50)为313μg/mL,SI为11.43。EPS 25mg.kg-1.d-1灌胃后,小鼠存活率为40.0%,存活时间为9.82±1.87天,相比模型组实验动物存活时间和存活率均有提高。菌株初步鉴定为淡紫色拟青霉。结论:从海南红树林分离一株淡紫色拟青霉,其产生的EPS具有抗HSV-2活性,值得进一步研究。     

Attenuation of human immunodeficiency virus type 1 cytopathic effect by a mutation affecting the transmembrane envelope glycoprotein.

The cytopathic effects of human immunodeficiency virus type 1 (HIV-1) infection are specific for cells that express the CD4 viral receptor and consist of syncytium formation and single-cell lysis. Here we report that a mutation (517A) affecting the amino terminus of the HIV-1 gp41 transmembrane envelope glycoprotein resulted in a virus that was markedly less cytopathic than was wild-type HIV-1. In systems in which cell-to-cell transmission of HIV-1 occurred, the replication ability of the 517A virus was comparable with that of the wild-type virus. Even though the levels of viral protein expression, virion production, and interaction of the envelope glycoproteins with CD4 were similar for the 517A and wild-type viruses, both syncytium formation and single-cell lysis were attenuated for the 517A mutant virus. These results demonstrate that an envelope glycoprotein region important for mediating post-receptor binding events in cell membrane fusion is important for the induction of cytopathic effects by HIV-1. These results also indicate that levels of HIV-1 viral proteins or viral particles produced in infected cells are in themselves not sufficient to induce cytopathic effects.     

Characterization of Exopolysaccharides (EPSs) Obtained from Ligilactobacillus salivarius Strains and Investigation at the Prebiotic Potential as an Alternative to Plant Prebiotics at Poultry

In this study, it was aimed to reveal the potential of using exopolysaccharides (EPS) obtained from Ligilactobacillus salivarius as a prebiotic that regulates chicken intestinal microbiota. Characterization of EPS obtained from L. salivarius BIS312 (EPSBIS312) and BIS722 (EPSBIS722) strains was demonstrated by high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and size-exclusion chromatography (SEC) analyses. It was determined that the molecular weight of both EPS is in the range of 10⁴–10⁶ Daltons, and there are 4 types of monomers in their structure. Anti-biofilm and anti-quorum sensing effects of EPSBIS312 and EPSBIS722 were determined. EPSBIS312 and EPSBIS722 showed a strong anti-biofilm effect on Enterococcus faecalis ATCC 29212, Staphylococcus aureus EB-1, and Escherichia coli ATCC 11229. The anti-quorum sensing study revealed that the EPSBIS722 had a higher effect than the EPSBIS312. The effect of different concentrations of EPS (2.5%, 5%, 10%) on lactobacilli growth stimulator (LGS) was evaluated. The highest LGS was promoted at 10% concentration while the lowest LGS was promoted at 2.5% concentration by EPSBIS722. In addition, adhesion abilities of EPSBIS312 and EPSBIS722 in HT-29 colorectal adenocarcinoma cell line were tested. EPSs significantly increased the ability to adhere to HT-29 cells. The characterized EPSs may be an alternative to plant prebiotics such as inulin at poultry.

     

Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis.

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.     

Cytopathic killing of peripheral blood CD4(+) T lymphocytes by human immunodeficiency virus type 1 appears necrotic rather than apoptotic and does not require env

An important unresolved issue of AIDS pathogenesis is the mechanism of human immunodeficiency virus (HIV)-induced CD4(+) T-lymphocyte destruction. We show here that HIV type 1 (HIV-1) exerts a profound cytopathic effect upon peripheral blood CD4(+) T lymphocytes that resembles necrosis rather than apoptosis. Necrotic cytopathology was found with both laboratory-adapted strains and primary isolates of HIV-1. We carefully investigated the role of env, which has been previously implicated in HIV cytopathicity. HIV-1 stocks with equivalent infectivity were prepared from constructs with either an intact or mutated env coding region and pseudotyped with the glycoprotein of vesicular stomatitis virus (VSV-G) so that the HIV envelope was not rate-limiting for infection. Infected Jurkat T cells died whether or not env was intact; however, the expression of env accelerated death significantly. The accelerated death was blocked by protease inhibitors, indicating that it was due to reinfection by newly produced virus in env(+) cultures. Accordingly, we found no disparity in kinetics in CD4(lo) Jurkat cells. In highly infected peripheral blood T cells, profound necrosis occurred equivalently with both env(+) and env(-) stocks of HIV-1. We also found that HIV-1 cytopathicity was undiminished by the absence of nef. However, viral stocks made by complementation or packaging of HIV-1 genomes with the natural protein-coding sequences replaced by the green fluorescent protein were highly infectious but not cytopathic. Thus, env can accelerate cell death chiefly as an entry function, but one or more viral functions other than env or nef is essential for necrosis of CD4(+) T cells induced by HIV-1.     

Cytopathicity of human immunodeficiency virus type 2 (HIV-2) in human lymphoid tissue is coreceptor dependent and comparable to that of HIV-1

Epidemiological studies have shown that human immunodeficiency virus type 2 (HIV-2) is markedly less pathogenic than HIV-1 in vivo. Individuals infected with HIV-2 exhibit a remarkably slow rate of disease development, and these clinical properties have been attributed presumptively to an "attenuated" phenotype of HIV-2 itself. Here, we investigated the impact of coreceptor usage on the cytopathicity of HIV-2 and compared its pathogenic potential with that of HIV-1 in a unique human lymphoid histoculture model. We found that HIV-2 strains, as well as closely related simian immunodeficiency viruses (SIV), displayed mildly or highly aggressive cytopathic phenotypes depending on their abilities to use the coreceptor CCR5 or CXCR4, respectively. A side-by-side comparison of primary X4 HIV-1 and HIV-2 strains revealed similar, high degrees of cytopathicity induced by both HIV types. Furthermore, we found that HIV-2 coreceptor specificity for CCR5 and CXCR4 determined the target cell population for T-cell depletion in lymphoid tissue. Finally, utilization of the alternate coreceptors BOB and Bonzo did not significantly increase the cytopathic properties of HIV-2. These findings demonstrate that coreceptor preference is a key regulator of target cell specificity and the cytopathic potential of HIV-2, with indistinguishable rules compared with HIV-1. Moreover, HIV-2 strains are not characterized by an intrinsically lower cytopathicity than HIV-1 strains. Therefore, direct cytopathic potential per se does not explain the unique behavior of HIV-2 in people, highlighting that other unknown factors need to be elucidated as the basis for their lesser virulence in vivo.     

Effect of cacao husk extract on human immunodeficiency virus infection

A sodium hydroxide extract from cacao husk inhibited the cytopathic effect of human immunodeficiency virus type 1 (HIV-1) against HTLV-1-transformed T-cell lines MT-2 and MT-4. It also inhibited syncytium formation between HIV-infected and uninfected lymphoblastoid T-cell line, MOLT-4. The anti-HIV activity was concentrated by membrane filter fractionation to a fraction with molecular weight of 100-300 KDa. Anti-HIV activity of the extract was attributable to interference with the virus adsorption, rather than to inhibition of the virus replication after adsorption.