The role of upstream U3 sequences in the pathogenesis of simian immunodeficiency virus-induced AIDS in rhesus monkeys

The nef reading frame overlaps about 70% of the U3 region of the 3' long terminal repeat (LTR) in primate lentiviruses. We investigated the functional role of these overlapping U3 sequences by analyzing the properties of three mutant forms of the pathogenic SIVmac239 clone. In mutant UScon, 90 of 275 bp in the upstream sequences (US) of U3 were changed in a conservative fashion without changing the predicted nef coding sequence. In mutant USnon, 101 of 275 bp in this region were changed in a nonconservative fashion, again without changing the predicted nef coding sequence. In mutant delta US, 275 bp in this region were deleted. Full-size, immunoreactive nef protein was synthesized in cells infected with the UScon and USnon mutants. The USnon and delta US mutants replicated with similar kinetics and to similar extents as wild-type, parental SIVmac239 in primary rhesus monkey peripheral blood mononuclear cell (PBMC) cultures. The UScon mutant replicated with slightly delayed kinetics in rhesus monkey PBMC cultures. In the CEMx174 cell line, the delta US mutant replicated similarly to the wild type, but the UScon and USnon mutants replicated with significantly delayed kinetics. Analysis of LTR-driven chloramphenicol acetyltransferase (CAT) activity and the effects of 5-azacytidine on virus replication suggested that the growth defect of the point mutants in CEMx174 cells was due in whole or in part to the introduction of multiple CG methylation sites in proviral DNA. Rhesus monkeys were experimentally infected with the UScon and USnon mutants, and the characteristics of the infection were compared with those of the parental SIVmac239. Analysis of the levels of plasma antigenemia, virus load, and CD4+ cells in PBMC revealed no decreased virulence of the mutant viruses. Analysis of lymph node biopsies taken from animals that received mutant viruses revealed histologic changes and levels of virus expression indistinguishable from those of the wild type. Furthermore, the wild-type behavior of the mutant viruses in rhesus monkeys occurred without any specific reversional events through at least 20 weeks of infection. These results, and the recent results of Kirchhoff et al. (F. Kirchoff, H. W. Kestler III, and R. C. Desrosiers, J. Virol. 68:2031-2037, 1994), suggest that these upstream sequences in U3 are primarily or exclusively nef coding sequence.     

Effect of cyclosporine on immune responses in submaxillary lymph nodes of pituitary-grafted rats

This work was undertaken to analyze the interrelationships between prolactin and cyclosporine in affecting immune responsiveness in submaxillary lymph nodes. Male rats received an anterior pituitary graft within breast muscles on day 5, or under the kidney capsule, on day 30 or 60 of life. On day 70 (rats operated on day 5 or 30) or on day 100 (rats operated on day 60) animals were injected with Freund's complete adjuvant and cyclosporine (5 mg/kg for 5 days), and were killed 2 days after immunization. Natural killer (NK) activity in submaxillary lymph node decreased in neonatally pituitary-grafted rats and increased in rats grafted on day 30 or 60, as did lymph node cellularity. Lipopolysaccharide (LPS)- and concanavalin A (ConA)-induced proliferation diminished in lymph nodes of rats grafted on day 30 or 60, respectively. Cyclosporine treatment diminished lymph node cell number and NK activity and increased the proliferative response to ConA. Cyclosporine depressive effect on lymph node cellularity was counteracted by the presence of a pituitary graft, as were the inhibition of NK activity and the stimulatory effect on ConA-induced cell proliferation. In pituitary-grafted rats, cyclosporine decreased submaxillary lymph node LPS-induced proliferation. Cyclosporine decreased the high circulating prolactin levels found in pituitary-grafted rats. The results are compatible with age-dependent, inhibitory and promoting activities of hyperprolactinemia on immune responses in lymph nodes, affected in a complex antagonistic and synergistic way by cyclosporine immunosuppression.     

New technique for lesser saphenous vein harvesting

A new technique using the Thompson self-retaining retractor system (Thompson Surgical Instruments, Inc, Traverse City, MI) to harvest lesser saphenous veins is presented. This modification, used in 10 patients undergoing redo myocardial revascularization, provided a rapid, comfortable, and convenient method for harvesting lesser saphenous veins.     

Electron paramagnetic resonance studies of cobalt-substituted angiotensin I-converting enzyme

The traditional treatment of high-flow vascular malformations consists of selective embolization, surgical removal, or a combination of both. Recurrence of the lesion and bleeding control are still the main problems, and the result of treatment is sometimes disappointing. We suggest treatment of these lesions with surgical ligation of the distal major feeding arteries followed by intravascular injection of a sclerosing agent (3% tetradecyl sulfate), and surgical excision and reconstruction when indicated. We have found this to be an effective treatment regimen. We present 14 cases of high-flow vascular malformations of the head and neck area treated with this approach, of which 4 cases developed skin necrosis. Three of these 4 cases of skin necrosis were later treated with skin grafting and, in 1 case, an upper arm skin tube flap was used for nasal tip reconstruction. Three cases underwent delayed reconstruction using tissue expanders. From a symptomatic and aesthetic point of view, preliminary satisfactory results were obtained. We feel that this approach is a good option for treating difficult, high-flow vascular malformations.     

Teleradiology by two different concepts. Technical note

Two different teleradiology concepts are described. Their advantages, disadvantages and costs are discussed.     

Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.     

Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids

The present study is intended to demonstrate the application of impedance spectroscopy to two very different fields of biophysical research. The core component of our measuring setup is a self-constructed continuous wave impedance spectrometer together with special measuring chambers which are individually designed for the systems under investigation. We directed our attention towards: i) the investigation of solid supported lipid bilayers in general, especially systems which are suitable for protein reconstitution such as dimethyldioctadecylammonium bromide (DODAB) immobilized onto a gold electrode, precovered with a negatively charged monolayer of 3-mercaptopropionic acid. Impedance spectroscopy allows to study the stability, the thickness and the electrode coverage of those artificial membranes as well as the observation of ion transport mediated by ionophores like gramicidin D incorporated into a DODAB-bilayer. ii) The characterization of the passive electrical properties of epithelial and endothelial cell monolayers in general and especially the determination of their transepithelial (transendothelial) electrical resistances as a measure for epithelial barrier function. From impedance spectra, as reported here, we are able to follow the formation and modulation of cell layer permeability to small ions.     

EFFECT OF CHLORINE TREATMENT ON CUT WATER CRESS AND ONION

Chlorine treatment was evaluated for cut vegetables to reduce microbial populations and improve keeping quality. Water cress and onion were selected because they are representative vegetables having high potential for processing into cut prepared products. Cut water cress had a high initial microbial contamintion of 10^7.5 cfu/g, while cut onion had only 10^1.7 cfu/g. The cut produce was treated by soaking in chlorine solutions of different concentrations at 25C for 1 min. Treatment with ≤100 ppm chlorine effectively reduced the microbial load of the produce without significant quality losses. High concentrations of chlorine resulted in greater microbial proliferation after 7 days, ascorbic acid destruction and significant color change in stored cut vegetables. The effectiveness of chlorine treatment was limited to short-term storage of precut vegetables, and did not provide extended shelf-life.     

Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.