Site-specific mutations in HIV-1 gp41 reveal a correlation between HIV-1-mediated bystander apoptosis and fusion/hemifusion

The loss of CD4(+) T cells in HIV-1 infections is hypothesized to be caused by apoptosis of bystander cells mediated by cell surface-expressed HIV-1 Env glycoprotein. However, the mechanism by which Env mediates this process remains controversial. Specifically, the role of HIV-1 gp120 binding to CD4 and CXCR4 versus the fusion process mediated by gp41 remains unresolved. Env-induced apoptosis in bystander cells has been shown to be gp41-dependent and correlates with the redistribution of membrane lipids between Env-expressing cells and target cells (hemifusion). Using a rational mutagenesis approach aimed at targeting Env function via the gp41 subunit, we examined the role of HIV gp41 in bystander apoptosis. A mutation in the fusion domain of gp41 (V513E) resulted in a fusion-defective Env that failed to induce apoptosis. A mutation in the gp41 N-terminal helix (G547D) reduced cell fusion capacity and apoptosis; conversely, an Env mutant with a deletion of the gp41 cytoplasmic tail (Ct Del) enhanced both cell-to-cell fusion and apoptosis. Most significantly, an Env mutant containing a substitution in the loop region of gp41 (D589L) mediated transfer of lipids (hemifusion) to bystander cells but was defective in cell-to-cell and to a lesser degree virus-to-cell fusion. This mutant was still able to induce apoptosis in bystander cells. Hence, we have provided the first direct evidence that gp41-mediated hemifusion is both required and sufficient for induction of apoptosis in bystander cells. These results may help to explain the mechanism of HIV-1 Env-induced T cell depletion.     

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe–S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N–S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.     

Clinical Significance of Circulating Serum Levels of sCD95 and TNF-α in Cytoprotection of Cervical Cancer

Background:This study correlates the serum levels of sCD95 & TNF-α with a simple cell-based assay to evaluate the capacity of the serum sample to induce apoptosis in Jurkat cells. Interlinking of these parameters can be explored to design a minimum invasive diagnostic strategy for cervical cancer (CC).Methods:Sera samples were assessed to induce apoptosis in Jurkat cells through FACS. Serum levels of sCD95 and TNF-α were measured by ELISA. JNK phosphorylation was evaluated in sera incubated Jurkat cells. Data was scrutinized through statistical analysis.Results:Significantly higher serum levels of sCD95 and lower TNF-α levels were observed in CC patients; their sera samples inhibited induction of apoptosis in Jurkat cells through reduced JNK phosphorylation. Statistical analysis linked these three parameters for the early screening of CC.Conclusion:Distinct sera levels of sCD95 & TNF-α in CC patients showed an anti-apoptotic effect, which can be considered for early detection of CC.Key Words: Apoptosis, sCD95, Jurkat Cells, Tumor Necrosis Factor-alpha, Uterine Cervical Neoplasms     

Comparative studies on kinetics of inhibition of protein synthesis in intact cells by ricin and conjugate of ricin B-chain with momordin

Cofilin is an actin-binding protein of low molecular weight which is widely distributed in eukaryotes and is deeply involved in the dynamics of actin assembly in the cytoplasm. The actin-binding ability of cofilin is inhibited by inositol phosphates (PIP2), and the PIP2- and actin-binding site(s) has been localized in residues W104 - M115 of the cofilin primary sequence (Yonezawa et al. 1991). In the present study, in order to further clarify the functional domains in cofilin molecule, we constructed expression vectors containing cDNAs of different size with deletion at the 3-region of the open reading frame. The truncated cofilin molecules produced in E. coli were purified and examined for their actin-binding and PIP2-binding ability. We found that the truncated cofilin molecule without C-terminal residues #100-#166 including the previously-described actin-binding site could be cross-linked with actin by EDC, a zero-length cross-linker. In addition, these truncated peptides as well as synthetic peptides corresponding to the N-terminal sequence of cofilin suppressed the inhibitory action of PIP2 on actin-cofilin interaction. These results strongly suggest that additional actin- and PIP2-binding sites exist in the N-terminal region of cofilin.     

Immunocontraceptive potential of recombinantly expressed minimized chicken riboflavin carrier protein (mini-RCP) in rodents

Chicken riboflavin carrier protein (RCP; 219 AA) harbours four linear epitopes, constituted by the peptide residues 3-23, 64-83, 130-147 and 200-219. Antibodies to these sequences bioneutralize maternal RCP and provide protection from pregnancy in rodents. In order to overcome the major histocompatibility complex-dependent variability in immune response often encountered with use of single peptides for vaccination in genetically outbred populations, we have assembled a novel synthetic gene, incorporating in tandem the nucleotide sequences coding for all the four neutralizing epitopes of chicken RCP and expressed in Escherichia coli. The gene product, mini-RCP has been characterized for its immunogenic properties and contraceptive potential in rodents. Immunization of rabbits and rats led to generation of antibodies against individual peptide components, as determined by enzyme-linked-immunosorbent assay (ELISA). However, immunized rats carried pregnancy to term and delivered healthy offsprings. Antisera from these rats exhibited decreased affinity of binding to the native protein. These findings suggest that the prospects of covalently-linked epitope peptides need to be cautiously evaluated during the design and development of peptide-based vaccines.     

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.     

Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa

Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.     

Contrasting use of CCR5 structural determinants by R5 and R5X4 variants within a human immunodeficiency virus type 1 primary isolate quasispecies

Macrophagetropic R5 human immunodeficiency virus type 1 (HIV-1) isolates often evolve into dualtropic R5X4 variants during disease progression. The structural basis for CCR5 coreceptor function has been studied in a limited number of prototype strains and suggests that R5 and R5X4 Envs interact differently with CCR5. However, differences between unrelated viruses may reflect strain-specific factors and do not necessarily represent changes resulting from R5 to R5X4 evolution of a virus in vivo. Here we addressed CCR5 domains involved in fusion for a large set of closely related yet functionally distinct variants within a primary isolate swarm, employing R5 and R5X4 Envs derived from the HIV-1 89.6(PI) quasispecies. R5 variants of 89.6(PI) could fuse using either N-terminal or extracellular loop CCR5 sequences in the context of CCR5/CXCR2 chimeras, similar to the unrelated R5 strain JRFL, but R5X4 variants of 89.6(PI) were highly dependent on the CCR5 N terminus. Similarly, R5 89.6(PI) variants and isolate JRFL tolerated N-terminal CCR5 deletions, but fusion by most R5X4 variants was markedly impaired. R5 89.6(PI) Envs also tolerated multiple extracellular domain substitutions, while R5X4 variants did not. In contrast to CCR5 use, fusion by R5X4 variants of 89.6(PI) was largely independent of the CXCR4 N-terminal region. Thus, R5 and R5X4 species from a single swarm differ in how they interact with CCR5. These results suggest that R5 Envs possess a highly plastic capacity to interact with multiple CCR5 regions and support the concept that viral evolution in vivo results from the emergence of R5X4 variants with the capacity to use the CXCR4 extracellular loops but demonstrate less-flexible interactions with CCR5 that are strongly dependent on the N-terminal region.     

Fatty acid regulation of liver X receptors (LXR) and peroxisome proliferator-activated receptor alpha (PPARalpha ) in HEK293 cells

Fatty acids bind to and regulate the activity of peroxisome proliferator-activated (PPAR) and liver X receptors (LXR). However, the role lipid metabolism plays in the control of intracellular fatty acid ligands is poorly understood. We have identified two strains of HEK293 cells that display differences in fatty acid regulation of nuclear receptors. Using full-length and Gal4-LBD chimeric receptors in functional assays, 20:4,n6 induced PPARalpha activity approximately 2.2-fold and suppressed LXRalpha activity by 80% (ED50 approximately 25-50 microm) in HEK293-E (early passage) cells but had no effect on PPARalpha or LXRalpha receptor activity in HEK293-L (late passage) cells. LXRbeta was insensitive to fatty acid regulation in both HEK293 strains. Metabolic labeling studies using [14C]20:4,n6 (at 100 microm) indicated that the uptake of 20:4,n6 and its assimilation into triacylglycerol, diacylglycerol, and polar lipids revealed no difference between the two strains. Such treatment increased total cellular 20:4,n6 ( approximately 11-fold) and its elongation product, 22:4,n6 ( approximately 3.6-fold), within 6 h. Non-esterified 20:4,n6 and 22:4,n6 represented 相似文献