Integration of foreign DNA and its consequences in mammalian systems

The insertion of foreign DNA into the genomes of established cells and organisms and the consequences of this integration event are of significance for viral oncology, reverse genetics, transgenic organisms, human somatic gene therapy and evolution. This review summarizes recent experimental findings and focuses on the alteration of cellular DNA methylation at regions remote from the site of insertion. We also discuss experimental data demonstrating that foreign DNA ingested by mice is not completely degraded in their gastrointestinal tracts; fragments of this DNA have been found to be covalently linked to DNA with 70% homology to the mouse IgE receptor gene.     

Germany’s journey toward 14 Tesla human magnetic resonance

Multiple sites within Germany operate human MRI systems with magnetic fields either at 7 Tesla or 9.4 Tesla. In 2013, these sites formed a network to facilitate and harmonize the research being conducted at the different sites and make this technology available to a larger community of researchers and clinicians not only within Germany, but also worldwide. The German Ultrahigh Field Imaging (GUFI) network has defined a strategic goal to establish a 14 Tesla whole-body human MRI system as a national research resource in Germany as the next progression in magnetic field strength. This paper summarizes the history of this initiative, the current status, the motivation for pursuing MR imaging and spectroscopy at such a high magnetic field strength, and the technical and funding challenges involved. It focuses on the scientific and science policy process from the perspective in Germany, and is not intended to be a comprehensive systematic review of the benefits and technical challenges of higher field strengths.

     

Transcription of the genome of adenovirus type 12. IV. Maps of stable late RNA from productively infected human cells

From human KB cells productively infected with adenovirus type 12, mRNA and stable nuclear RNA were isolated late (42 h) after infection. Using restriction endonuclease fragments of adenovirus type 12 DNA, mRNA and stable nuclear RNA sequences were mapped on the viral genome. Late after infection, preferentially the r (= rightward) strand is transcribed into stable nuclear RNA, whereas the l (= leftward) strand is expressed only to a minor extent. Adenovirus type 12-specific mRNA originates from the following sections on the viral genome: 0 to 0.11, 0.18 to 0.20, 0.27 to 0.49, 0.56 to 0.63, 0.68 to 0.84, and 0.89 to 0.92 fractional length units on the r strand and 0.11 to 0.16, 0.22 to 0.27, 0.50 to 0.54, 0.62 to 0.66, 0.855 to 0.865, and 0.93 to 1.0 fractional length units on the l strand. Self-complementary viral RNA isolated at 42 h postinfection anneals to 70 to 80% of each strand of the viral genome.     

Potent CD14-mediated signalling of human leukocytes by Escherichia coli can be mediated by interaction of whole bacteria and host cells without extensive prior release of endotoxin

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.     

Role of tachykinins in ozone-induced acute lung injury in guinea pigs

To examine the hypothesis that the acute reversible changes caused by ozone (O3) exposure are mediated by tachykinin release, guinea pigs were depleted of tachykinins by use of repeated capsaicin (CAP) injections before O3 exposure in an attempt to prevent O3-induced functional changes. Unexpectedly, CAP pretreatment caused divergent results in the functional responses to O3. Ventilatory measurements obtained from CAP-pretreated O3-exposed (CAP-O3) animals were exacerbated rather than diminished compared with the effects of O3 alone. Similarly, lavage fluid protein accumulation was enhanced in the CAP-O3 group compared with the O3-exposed group. In better agreement with our initial hypothesis, the CAP-O3 group was less responsive than the O3-exposed animals to histamine aerosol challenge. Additionally, Evans blue dye accumulation, a hallmark of tachykinin release, was increased in O3-exposed animals and was partially blocked in the CAP-O3 group. These data suggest that tachykinin-containing sensory fibers are unlikely to mediate the acute effects of O3 exposure on tidal breathing and lavage fluid protein accumulation but may play a role in causing post-O3 airway hyperreactivity and protein extravasation into the trachea.     

Leukotriene B4 and interleukin-8 in human immunodeficiency virus-related pulmonary disease

STUDY OBJECTIVE: To investigate the pathogenesis of lung injury in Pneumocystic carinii pneumonia and nonspecific interstitial pneumonitis (NIP), common pulmonary complications of human immunodeficiency virus (HIV) infection. The efficacy of corticosteroid therapy in P carinii pneumonia and the observation that bronchoalveolar lavage (BAL) neutrophilia predicts a poor prognosis support the premise that the lung injury of P carinii pneumonia is due to the host's inflammatory response to the infection. DESIGN: In vitro measurements on previously collected BAL fluid samples. SETTING: The Clinical Center of the National Institutes of Health, a research hospital and tertiary care referral center. PATIENTS: Five normal volunteers, 5 asymptomatic HIV-positive patients, 10 HIV-positive patients with NIP (5 asymptomatic and 5 with respiratory symptoms), and 19 HIV-positive patients with P carinii pneumonia. MEASUREMENTS AND RESULTS: BAL leukotriene B4 (LTB4), interleukin 8 (IL-8), and phospholipase A2 (PLA2) were measured. IL-8 and PLA2 were elevated in patients with P carinii pneumonia, and IL-8 correlated with BAL fluid absolute neutrophil count. LTB4, IL-8, and PLA2 levels were elevated in patients with NIP; LTB4 and PLA2 levels correlated with absolute neutrophil count, and IL-8 correlated with alveolar-arterial oxygen pressure difference. IL-8 was elevated in the asymptomatic HIV-positive patients, and there was a trend toward elevation of PLA2 in this group. CONCLUSION: IL-8 appears to play a role in the pathogenesis of lung injury in P carinii pneumonia and may be the principal neutrophil chemotaxin in this disease; PLA2 may also be involved in the pathogenesis of P carinii pneumonia. Both LTB4 and IL-8 may be involved in the recruitment of neutrophils and subsequent lung injury of NIP. These data suggest that there are varying mechanisms by which inflammatory cells are recruited to the lung in different HIV-related lung diseases.     

Pain unit director: Role issues for health psychologists.

The increasing involvement of psychologists in the treatment of chronic pain has led to the emergence of psychologists as directors of multidisciplinary pain treatment programs. Although the role of pain unit director offers many opportunities to psychologists, several potential problems exist. Relationships with physicians must be carefully addressed; adequate administrative support must be secured; and the requisite clinical privileges must be sanctioned. Without such interprofessional and administrative groundwork, psychologists will have difficulty functioning within a medically dominated health care system. (16 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Transcription of the genome of adenovirus type 12. III. Maps of stable RNA from productively infected human cells and abortively infected and transformed hamster cells

The adenovirus type 12-specific mRNA and the stable nuclear RNA from productively infected KB cells, early postinfection, from abortively infected BHK-21 cells, and from the adenovirus type 12-transformed hamster lines T637 and HA12/7 have been mapped on the genome of adenovirus type 12. The intact separated heavy (H) and light (L) strands of adenovirus type 12 DNA have been used to determine the extent of complementarity of the mRNA or nuclear RNA from different cell lines to each of the strands. More precise map positions have been obtained by the use of the H and L complements of the fragments of adenovirus type 12 DNA which were produced with the EcoRI and BamHI restriction endonucleases. The results of the mapping experiments demonstrate that the mRNA's isolated early from productively and abortively infected and from two lines of transformed cells are derived from the same or similar regions of the adenovirus type 12 genome. The map positions on the adenovirus type 12 genome for the mRNA from the cell lines as indicated correspond to regions located approximately between 0 and 0.1 and 0.74 and 0.88 fractional length units on the L strand and to regions between 0.63 and 0.74 and 0.89 and 1.0 fractional length units on the H strand. The HA12/7 line lacks mRNA complementary to the region between 0.74 and 0.88 fractional length units on the L strand. Similar data are found for the nuclear RNA, except that the regions transcribed are more extensive than those observed in mRNA. The polarity of the H strand has its 3'-end on the right terminus in the EcoRI A fragment, and the L strand has its 3'-end on the left terminus in the EcoRI C fragment. Thus, the H strand is transcribed from right to left (1 = leftward strand); and the L strand is transcribed from left to right (r = rightward strand). The designations H and L refer to the relative heavy and light densities of the two strands in polyuridylic-polyguanylic acid-CsCl density gradients. The EcoRI C-H and D-H complements have been shown to be part of the intact L strand; thus, there is a "reversal in heaviness" on the left terminus of the viral DNA.     

Rapid protein sequencing by tandem mass spectrometry and cDNA cloning of p20-CGGBP. A novel protein that binds to the unstable triplet repeat 5'-d(CGG)n-3' in the human FMR1 gene

The autonomous expansion of the unstable 5'-d(CGG)n-3' repeat in the 5'-untranslated region of the human FMR1 gene leads to the fragile X syndrome, one of the most frequent causes of mental retardation in human males. We have recently described the isolation of a protein p20-CGGBP that binds sequence-specifically to the double-stranded trinucleotide repeat 5'-d(CGG)-3' (Deissler, H., Behn-Krappa, A., and Doerfler, W. (1996) J. Biol. Chem. 271, 4327-4334). We demonstrate now that the p20-CGGBP can also bind to an interrupted repeat sequence. Peptide sequence tags of p20-CGGBP obtained by nanoelectrospray mass spectrometry were screened against an expressed sequence tag data base, retrieving a clone that contained the full-length coding sequence for p20-CGGBP. A bacterially expressed fusion protein p20-CGGBP-6xHis exhibits a binding pattern to the double-stranded 5'-d(CGG)n-3' repeat similar to that of the authentic p20-CGGBP. This novel protein lacks any overall homology to other known proteins but carries a putative nuclear localization signal. The p20-CGGBP gene is conserved among mammals but shows no homology to non-vertebrate species. The gene encoding the sequence for the new protein has been mapped to human chromosome 3.     

Mouse palatal width growth rates as an "at risk" factor in the development of cleft palate induced by hypervitaminosis A

In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86 + C1300, expressing a high level of mouse CD86 on the cell surface. While CD86 + C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86 + C1300-rejecting mice with CD86 + C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86 + C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86 + C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86 + C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors.