Results Following HIV infection of SVGA reporter cells, very limited infection was detected. Further, no receptor or coreceptors except CXCR4 were present on astrocytes. To see further if viral replication is blocked in astrocytes, we infected PFA or SVGA cells with VSV pseudotyped NL4-3. High levels of p24 and robust LTR-GFP or LTR-gagGFP activation were seen. VSV-HIV infected reporter cells never lost green fluorescence and green cells were negative despite of continuous low Tat and Rev production. To rule out If Tat and Rev expression were from circular unintegrated HIV-DNA, Alu PCR revealed integration of viral DNA. Further, Tat or TNF-a reactivated the latent HIV in astrocytes and the latent HIV-SVGA cells upon co-culture transmitted the infection to Jurkat cells.
A genetic progression model for head and neck squamous cell carcinoma (HNSC) has been established and implies the presence of transcriptional dysregulation as a consequence of accumulation of genetic alterations. Although expression array data have been provided for HNSC, the timing of transcriptional dysregulation in the progression from normal mucosa to dyplastic epithelium to invasive HNSC has not been described. Here, we describe a transcriptional progression model of HNSC.Expression arrays representing >12,000 genes and expressed sequence tags were used to examine malignant lesions (M), premalignant lesions (PM), distant, histopathologically normal mucosa from patients with premalignant or malignant lesions (MN), and normal mucosa from the upper aerodigestive tract of patients with noncancer diagnoses (N). Significance analysis of microarrays, hierarchical clustering, and principal components analysis was used to identify genes with differential expression patterns.Using a false discovery rate of <5% for significance analysis of microarray, the M group revealed 965 up-regulated and 1106 down-regulated genes relative to the N group. The PM group demonstrated 108 up-regulated and 226 down-regulated genes relative to the N group, whereas the M group demonstrated only 5 up-regulated and 13 down-regulated genes relative to the PM group. Both hierarchical cluster analysis and principal components analysis revealed a consistent separation between the N, PM, and M groups, with a closer association between the PM and M groups. To provide independent validation of the microarray data, quantitative reverse transcription-PCR was performed for a significantly up-regulated gene, integrin alpha 6, correlating well with microarray data (linear regression analysis, P < 0.0001).Similarly to the genetic progression model of HNSC, this transcriptional model shows that the majority of alterations occurs before the development of malignancy and identifies key targets of transcriptional dysregulation during progression from a normal to a premalignant state and from a premalignant to a malignant state.
Candida glabratais an opportunistic pathogen in humans, responsible for approximately 20% of disseminated candidiasis. Candida glabrata's ability to adhere to host tissue is mediated by GPI-anchored cell wall proteins (GPI-CWPs); the corresponding genes contain long tandem repeat regions. These repeat regions resulted in assembly errors in the reference genome. Here, we performed a de novo assembly of the C. glabrata type strain CBS138 using long single-molecule real-time reads, with short read sequences (Illumina) for refinement, and constructed telomere-to-telomere assemblies of all 13 chromosomes. Our assembly has excellent agreement overall with the current reference genome, but we made substantial corrections within tandem repeat regions. Specifically, we removed 62 genes of which 45 were scrambled due to misassembly in the reference. We annotated 31 novel ORFs of which 24 ORFs are GPI-CWPs. In addition, we corrected the tandem repeat structure of an additional 21 genes. Our corrections to the genome were substantial, with the length of new genes and tandem repeat corrections amounting to approximately 3.8% of the ORFeome length. As most corrections were within the coding regions of GPI-CWP genes, our genome assembly establishes a high-quality reference set of genes and repeat structures for the functional analysis of these cell surface proteins.
Abnormal vascular phenotypes have been implicated in neuropathologies ranging from Alzheimer's disease to brain tumors. The development of transgenic mouse models of such diseases has created a crucial need for characterizing the murine neurovasculature. Although histologic techniques are excellent for imaging the microvasculature at submicron resolutions, they offer only limited coverage. It is also challenging to reconstruct the three-dimensional (3D) vasculature and other structures, such as white matter tracts, after tissue sectioning. Here, we describe a novel method for 3D whole-brain mapping of the murine vasculature using magnetic resonance microscopy (μMRI), and its application to a preclinical brain tumor model. The 3D vascular architecture was characterized by six morphologic parameters: vessel length, vessel radius, microvessel density, length per unit volume, fractional blood volume, and tortuosity. Region-of-interest analysis showed significant differences in the vascular phenotype between the tumor and the contralateral brain, as well as between postinoculation day 12 and day 17 tumors. These results unequivocally show the feasibility of using μMRI to characterize the vascular phenotype of brain tumors. Finally, we show that combining these vascular data with coregistered images acquired with diffusion-weighted MRI provides a new tool for investigating the relationship between angiogenesis and concomitant changes in the brain tumor microenvironment.
We have studied the molecular patterns formed in Langmuir–Blodgett (LB) films of semifluorinated alkanes CnF2n+1CmH2m+1 (FnHm diblocks, n = 6, 8, 10, m = 14, 16, 18, 20) by atomic force microscopy. All the compounds investigated formed surface micelles whose shape and dimensions varied with the molecular structure of the FnHm diblocks. Except for F6H16 and F8H14, which produced exclusively circular micelles, all of the other diblocks also formed elongated micelles that coexisted with circular micelles. The mean diameter of the circular micelles increased with the length of the hydrogenated segment of the diblocks. By contrast, increasing the length of the fluorinated segments did not have any detectable effect on the diameter. The elongated micelles became more numerous and longer for longer FnHm (both the length of the hydrogenated and of the fluorinated segments had a strong effect). The surface pressure of transfer had an influence on the morphology of the elongated micelles, the latter becoming fewer and shorter for higher surface pressures. A detailed X-ray reflectivity study conducted on F8H16 LB films showed that the hydrogenated segments are directed towards the silicon wafer, while the fluorinated segments point outwards toward the air. A disc-like model is proposed for the surface micelles.
Non-Hodgkin's (NHL) B cell lymphomas are growth-inhibited by ligation of their CD40 molecules. This inhibition is not absolute in that ≈ 50% of the cells are not inhibited. We conducted studies to see if other signals that have been reported to inhibit B cell lymphoma growth could be used in combination with anti-CD40 signaling to completely inhibit growth. Ligation of surface immunoglobulin (Ig), CD19, CD20, CD37 or CD95 with soluble antibody did not affect growth of the panel of NHL cells examined. Ligation of CD20, CD19 or CD95 was inhibitory for some NHL cell lines if the primary antibody was crosslinked with a secondary antibody. Combining anti-CD40 with anti-CD19, anti-CD20, or anti-Ig resulted in increased inhibition past that produced by anti-CD40 alone. The additive effect of anti-CD40 and other antibodies to selected surface markers was not observed in all NHL cell lines. Crosslinking of CD95 was also growth inhibitory for the majority of the NHL, and when combined with anti-CD40 under conditions that afforded crosslinking of the two receptors, increased inhibition was seen in three of the NHL cell lines. We found that cAMP or sodium butyrate (NaB) were also effective at inhibiting growth of the NHL cells; this was a profound inhibition (approaching 100%) compared to the 50% inhibition seen with anti-CD40 treatment. The potential for anti-CD40 and either cAMP or NaB to be additive was tested and not found to be the case. The ability to inhibit proliferation of the NHL was very dynamic with some antibody combinations being either inhibitory for multiple cells, not having an effect at all, or in some cases being stimulatory. This suggests that the NHL may represent unique stages of B cells that might serve as a model system which could be developed to precisely categorize patient NHL.
Syntheses of eight novel A-ring cleaved oleanane and ursane analogs are described. These compounds were assessed for their ability to inhibit cell proliferation in NRP.152 prostate cells. Four A-ring cleaved derivatives showed significant activity; 5β-(1-methyl-2-ethyl)-10α-(3-aminopropyl)-des-A-urs-12-en-28-oic acid was the most active compound, (IC50, 0.3μM).
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