Mutagenesis analysis of the murine leukemia virus matrix protein: identification of regions important for membrane localization and intracellular transport

We have created two sets of substitution mutations in the Moloney murine leukemia virus (Mo-MuLV) matrix protein in order to identify domains involved in association with the plasma membrane and in incorporation of the viral envelope glycoproteins into virus particles. The first set of mutations was targeted at putative membrane-associating regions similar to those of the human immunodeficiency virus type 1 matrix protein, which include a polybasic region at the N terminus of the Mo-MuLV matrix protein and two regions predicted to form beta strands. The second set of mutations was created within hydrophobic residues to test for the production of virus particles lacking envelope proteins, with the speculation of an involvement of the membrane-spanning region of the envelope protein in incorporation into virus particles. We have found that mutation of the N-terminal polybasic region redirected virus assembly to the cytoplasm, and we show that tryptophan residues may also play a significant role in the intracellular transport of the matrix protein. In total, 21 mutants of the Mo-MuLV matrix protein were produced, but we did not observe any mutant virus particles lacking the envelope glycoproteins, suggesting that a direct interaction between the Mo-MuLV matrix protein and envelope proteins either may not exist or may occur through multiple redundant interactions.     

Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected Müller cells temporarily rescues injured retinal ganglion cells

In this study, we demonstrate that: (i) injection of an adenovirus (Ad) vector containing the brain-derived neurotrophic factor (BDNF) gene (Ad.BDNF) into the vitreous chamber of adult rats results in selective transgene expression by Müller cells; (ii) in vitro, Müller cells infected with Ad.BDNF secrete BDNF that enhances neuronal survival; (iii) in vivo, Ad-mediated expression of functional BDNF by Müller cells, temporarily extends the survival of axotomized retinal ganglion cells (RGCs); 16 days after axotomy, injured retinas treated with Ad.BDNF showed a 4.5-fold increase in surviving RGCs compared with control retinas; (iv) the transient expression of the BDNF transgene, which lasted approximately 10 days, can be prolonged with immunosuppression for at least 30 days, and such Ad-mediated BDNF remains biologically active, (v) persistent expression of BDNF by infected Müller cells does not further enhance the survival of injured RGCs, indicating that the effect of this neurotrophin on RGC survival is limited by changes induced by the lesion within 10-16 days after optic nerve transection rather than the availability of BDNF. Thus, Ad-transduced Müller cells are a novel pathway for sustained delivery of BDNF to acutely-injured RGCs. Because these cells span the entire thickness of the retina, Ad-mediated gene delivery to Müller cells may also be useful to influence photoreceptors and other retinal neurons.     

Microtiter assay for detecting Campylobacter spp. and Helicobacter pylori with surface gangliosides which bind cholera toxin

Campylobacter jejuni with Gm1 ganglioside in the core of its lipopolysaccharide has been associated with Guillain-Barré syndrome. Since this epitope may be of considerable pathophysiologic importance and since this ganglioside binds cholera toxin, a rapid screening assay to detect bacteria that bind cholera toxin as an indication of Gm1 on their surfaces was developed. In the assay, bacterial lawns were grown on agar plates, harvested with phosphate-buffered saline, boiled, and incubated with a standard concentration of cholera B subunit. Preparations from strains with Gm1 were observed to inhibit the binding of cholera B subunit to Gm1 in a microtiter enzyme-linked immunosorbent assay. By using this assay with two groups of strains, 37 positive strains were detected among the 197 tested. Species with positive isolates included C. jejuni, Campylobacter coli, and Helicobacter pylori. The assay is capable of testing large numbers of isolates and should prove useful in future clinical and epidemiological studies of bacteria with this epitope.     

Rapid transepithelial antigen transport in rat jejunum: impact of sensitization and the hypersensitivity reaction

BACKGROUND & AIMS: Intestine from sensitized rats develops a rapid secretory response to luminal antigen challenge that depends on activation of subepithelial mast cells. The aim of this study was to determine the timing and route of the transepithelial protein antigen transport. METHODS: Rats were sensitized to horseradish peroxidase (HRP). After 10-14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. RESULTS: Electron microscopy of tissue specimens fixed at 2 minutes (before mast cell activation) showed enhanced endocytic uptake of HRP in enterocytes of HRP-sensitized rats compared with ovalbumin-sensitized or saline-injected controls. At this time, HRP was distributed throughout epithelial cells and was already evident in the lamina propria. In contrast, HRP was restricted to the apical region of enterocytes in controls. At 30 minutes (after mast cell activation), in HRP-sensitized rats only, HRP was also located within tight junctions and the paracellular region between epithelial cells. Tissue conductance was increased in HRP-sensitized rats beginning 30 minutes after HRP addition and correlated with the overall flux of HRP across the tissue. CONCLUSIONS: The results show that specific sensitization enhances the initial uptake and transcytosis of antigen across intestinal epithelium. Subsequent to activation of mast cells, antigen transport is further enhanced by penetration through the paracellular pathway.     

Metacarpophalangeal joint dislocation: indications for open surgical reduction

In complex dislocations of the metacarpophalangeal joint, the volar plate is separated from the proximal phalanx and the metacarpal head is entrapped within surrounding tissue structures. These complex dislocations must be managed by open surgical reduction to reduce the dislocation and realign the volar plate. A 58-year-old male presented to the emergency department with a complex dislocation of the metacarpophalangeal joint of the left little finger, which was successfully treated by open reduction in the operating room. The indications for open reduction of metacarpophalangeal joint dislocations are reviewed.     

Regional blood flow during pulsatile cardiopulmonary bypass and after circulatory arrest in an infant model

BACKGROUND: Pulsatile perfusion systems have been proposed as a means of improving end-organ perfusion during and after cardiopulmonary bypass. Few attempts have been made to study this issue in an infant model. METHODS: Neonatal piglets were subjected to nonpulsatile (n = 6) or pulsatile (n = 7) cardiopulmonary bypass and 60 minutes of circulatory arrest. Cerebral, renal, and myocardial blood flow measurements were obtained at baseline, on bypass before and after circulatory arrest, and after bypass. RESULTS: Cerebral blood flow did not differ between groups at any time and was diminished equally in both groups after circulatory arrest. Renal blood flow was diminished in both groups during bypass but was significantly better in the pulsatile group than in the nonpulsatile group prior to, but not after, circulatory arrest. Myocardial blood flow was maintained at or above baseline in the pulsatile group throughout the study, but in the nonpulsatile group, it was significantly lower than baseline during CPB prior to circulatory arrest and lower compared with baseline and with the pulsatile group 60 minutes after CPB. CONCLUSIONS: Pulsatile bypass does not improve recovery of cerebral blood flow after circulatory arrest, may improve renal perfusion during bypass but does not improve its recovery after ischemia, and may have beneficial effects on myocardial blood flow during bypass and after ischemia compared with nonpulsatile bypass in this infant model.