Disruption of Helix-Capping Residues 671 and 674 Reveals a Role in HIV-1 Entry for a Specialized Hinge Segment of the Membrane Proximal External Region of gp41

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662–683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell–cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.     

p21 of Kirsten murine sarcoma virus is thermolabile in a viral mutant temperature sensitive for the maintenance of transformation.

We have recently described an intracellular protein, p21, in nonproducer cells transformed by either the Kirsten (Ki-MSV) or Harvey (Ha-MSV) strain of murine sarcoma virus (Shih et al., Virology, in press). The p21 is phosphorylated and has been shown to be coded for by either Ki-MSV or Ha-MSV. In this report, we compare the thermal stability of the newly synthesized [35S]methionine-labeled p21 in cells transformed by the wild-type Ki-MSV or by a mutant of Ki-MSV (ts 371) which is temperature sensitive in a viral function required for the maintenance of several properties of the transformed phenotype. The immunoprecipitability of the p21 coded for by the ts 371 Ki-MSV was markedly more thermolabile than the p21 of the wild-type Ki-MSV when the cell extracts are heated in vitro. The present finding suggests that the p21 is required for the maintenance of transformation induced by Ki-MSV.     

Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus

Various domains of hepatitis B surface antigen (HBsAg) are essential for the assembly and secretion of hepatitis D virus (HDV). This study investigated the influences of the levels and sequences of HBsAg of naturally occurring HBV variants on the assembly and secretion of HDV. Six hepatitis B virus (HBV)-producing plasmids (three genotype B and three genotype C) and six HBsAg expression plasmids that expressed various HBsAg levels were constructed from the sera of HDV-infected patients. These plasmids were cotransfected with six expression plasmids of HDV of genotype 1, 2, or 4 into the Huh-7 hepatoma cell line. Serum HBsAg and HBV DNA levels were correlated with HDV RNA levels and outcomes of chronic hepatitis D (CHD) patients. The secretion of genotype 1, 2, or 4 HDV generally correlated with HBsAg levels but not with HBV genotypes or HBV DNA levels. Swapping and residue mutagenesis experiments of HBsAg-coding sequences revealed that the residue Pro-62 in the cytosolic domain-I affects the assembly and secretion of genotype 2 and 4 HDV and not those of genotype 1. The pre-S2 N-terminal deletion HBV mutant adversely affects secretion of the three HDV genotypes. In patients, serum HDV RNA levels correlated with HBsAg levels but not with HBV DNA levels. Viremia of HDV or HBV correlated with poor outcomes. In conclusion, the assembly and secretion of HDV were influenced by the amounts and sequences of HBsAg. For an effective treatment of CHD, reduction of HBsAg production in addition to the suppression of HBV and HDV replication might be crucial.     

Adipose tissue growth and regression are regulated by angiopoietin-1

Adipose tissue is unique in its plasticity, capacity for vascular remodeling, and susceptibility to angiogenesis inhibitors. We hypothesize that these characteristics are enabled by maintaining relatively immature adipose vessels to facilitate vascular/tissue remodeling. We examined the vascular maturation regulators, angiopoietin-1, angiopoietin-2, and tie2 receptor, under different weight-modifying conditions. Adipocytes expressed angiopoietin-1, while adipose endothelial cells expressed angiopoietin-2 and tie2. Adipose tissue growth/regression were associated with decreased angiopoietin-1 mRNA and protein, and tie2 phosphorylation. Angiopoietin-2 and tie2 mRNA levels were stable. Angiopoietin-1 mRNA levels inversely correlated with the rates of change in body weight, independent of the direction (weight gain, loss) or etiology (TNP-470, leptin, and diet restriction) of the weight shift. Obese mice injected with ang1/pcDNA had reduced rates of weight gain and fat pad weights, regardless of the route of plasmid administration (subcutaneous, intramuscular, and intravenous). Thus, angiopoietin-1 may regulate adipose tissue growth, suggesting that vascular maturation alters tissue plasticity.     

JARID1B Expression Plays a Critical Role in Chemoresistance and Stem Cell-Like Phenotype of Neuroblastoma Cells

Neuroblastoma (NB) is a common neural crest-derived extracranial solid cancer in children. Among all childhood cancers, NB causes devastating loss of young lives as it accounts for 15% of childhood cancer mortality. Neuroblastoma, especially high-risk stage 4 NB with MYCN amplification has limited treatment options and associated with poor prognosis. This necessitates the need for novel effective therapeutic strategy. JARID1B, also known as KDM5B, is a histone lysine demethylase, identified as an oncogene in many cancer types. Clinical data obtained from freely-accessible databases show a negative correlation between JARID1B expression and survival rates. Here, we demonstrated for the first time the role of JARID1B in the enhancement of stem cell-like activities and drug resistance in NB cells. We showed that JARID1B may be overexpressed in either MYCN amplification (SK-N-BE(2)) or MYCN-non-amplified (SK-N-SH and SK-N-FI) cell lines. JARID1B expression was found enriched in tumor spheres of SK-N-BE(2) and SK-N-DZ. Moreover, SK-N-BE(2) spheroids were more resistant to chemotherapeutics as compared to parental cells. In addition, we demonstrated that JARID1B-silenced cells acquired a decreased propensity for tumor invasion and tumorsphere formation, but increased sensitivity to cisplatin treatment. Mechanistically, reduced JARID1B expression led to the downregulation of Notch/Jagged signaling. Collectively, we provided evidence that JARID1B via modulation of stemness-related signaling is a putative novel therapeutic target for treating malignant NB.     

Definition of the carbohydrate response element of the rat S14 gene. Evidence for a common factor required for carbohydrate regulation of hepatic genes.

The 5'-flanking region of the S14 gene from -4316 to +18 contains regulatory sequences responsible for activation of promoter activity in response to elevated carbohydrate metabolism in primary hepatocytes. To map these sequences, a series of constructs containing various internal deletions of the S14 5'-flanking sequence were assayed in primary hepatocytes. The region from -1601 to -1395 was found to be essential for this response. Comparison of the sequence of this S14 region to a region of the L-type pyruvate kinase gene that has been shown to mediate carbohydrate regulation (Thompson, K. S., and Towle, H. C. (1991) J. Biol. Chem. 266, 8679-8682) revealed a segment with 9 out of 10 identity. In both cases, this conserved sequence aligned with a DNase I footprint formed with hepatic nuclear extract. Oligonucleotides (approximately 30 base pairs) from either S14 or pyruvate kinase genes containing the conserved element bound to a hepatic nuclear factor(s) that gave identical complexes by mobility shift assay. Furthermore, these two oligonucleotides cross-competed for binding to the nuclear factor(s), suggesting that a common factor(s) binds to this conserved element. Reinsertion of the S14 oligonucleotide into an unresponsive S14 promoter construct restored the carbohydrate control. Moreover, this oligonucleotide could confer a glucose response when fused to a heterologous promoter. Thus, the S14 segment from -1457 to -1428 is a carbohydrate response element essential for the binding of nuclear factor(s) regulated by increased carbohydrate metabolism. This factor(s) may be common to the carbohydrate regulation of the S14 and pyruvate kinase genes.     

Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum

Cryptosporidium parvum is a waterborne pathogen that poses potential risk to drinking water consumers. The detection of Cryptosporidium oocysts, its transmissive stage, is used in the latest U.S. Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-Cryptosporidium sp. monoclonal antibody (MAb). However, FITC exhibits low resistance to photodegradation. This property will inevitably limit the detection accuracy after a short period of continuous illumination. In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection. In this study, QD605-streptavidin together with biotinylated MAb was used for C. parvum oocyst detection. The C. parvum oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above. The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 +/- 501 oocysts (mean +/- standard deviation) was determined to be 20 nM. The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (P > 0.126) when various C. parvum oocyst concentrations were used. The QD605 was highly photostable while the FITC intensity decreased to 19.5% +/- 5.6% of its initial intensity after 5 min of continuous illumination. The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water. This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting C. parvum oocysts with the advantage of having a high photobleaching threshold.     

Free proline levels in ulva (chlorophyta) in response to hypersalinity: elevated nacl in seawater versus concentrated seawater (Note)

Changes in free proline concentrations following exposure to elevated NaCl in 35 psu artificial seawater (ASW) (an imbalance in ion ratio) or concentrated ASW (constant ion ratio) were investigated in the intertidal green alga Ulva fasciata Delile. When exposed to 55 psu achieved by adding NaCl to 35 psu ASW, free proline concentrations increased at hour 3 and reached a plateau after 12 h, whereas free proline concentrations in response to 55 psu achieved by concentrating ASW were constant during the treatment period. It is likely that an imbalance in ion ratio is a cue for the induction of a moderate accumulation of free proline in U. fasciata .     

Growth of AKR T cell leukemia lymphoblasts in medium containing interleukin 2 (IL-2)

Long-term cloned mouse leukemic T cell lines were established in vitro using interleukin-2 (IL-2) conditioned media. The cell lines were tested for retention of both antigenic expression and tumorigenicity, as well as for growth characteristics. Several important findings resulted from these studies. A reliable method was developed for consistent success in the culturing and cloning of leukemic T cell lines. Cultured cells from IL-2-dependent lines were found to retain their original histocompatibility and differentiation antigen characteristics for up to 2 yr. Mortality patterns, comparing long-term cloned leukemic T cell lines with fresh AKR leukemia cells, showed that the cloned cells had greater tumorigenicity. An especially interesting finding was that cell lines established both from different mice and from a single organ of an individual mouse were heterogeneous with respect to antigenic makeup, cell growth kinetics, and tumorigenicity. Finally, because of their dependence on IL-2, the cloned tumor cell lines provided excellent index cells to quantify IL-2 activity.