Induction of accelerated feline immunodeficiency virus disease by acute-phase virus passage.

Development of feline immunodeficiency virus (FIV) infection in cats as a small animal model for lentiviral immunodeficiency disease has been hampered by the prolonged and variable disease course following experimental infection. To address this issue, we generated high-titer, unselected FIV stocks by pooling plasma from cats acutely infected with a subgroup C FIV isolate designated CABCpadyOOC (FIV-C-PGammer). Subsequent infection with this virus pool resulted in rapidly progressive, fatal disease in greater than 50% of infected cats. Accelerated FIV disease was characterized by rapid and progressive CD4+ T-cell loss, lymphadenopathy, weight loss, lymphoid depletion, and severe thymic atrophy. Mortality and rate of disease progression were affected by the age of each cat at infection and whether the virus source animal was in the acute or chronic stage of infection. The rapid FIV disease syndrome was consistently associated with systemic lymphoid depletion, clinical disease, and susceptibility to opportunistic infections, analogous to accelerated and/or terminal HIV-1 infection. The results of this study demonstrate that FIV infection is a valid small animal model for lentiviral immunodeficiency disease.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution.

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.     

Clinical trials of amniotic membranes in burn wound care

Four test conditions of increasing complexity were used to evaluate the clinical efficacy of amniotic membranes as biologic dressings on donor sites and burn wounds in children. These were the clean-skin donor-site wound, the uncontaminated shallow partial-thickness burn wound, the bed of freshly excised full-thickness wounds, and the granulating surface of colonized burn wounds. The rate of epithelialization under amniotic membranes was the same as that under 5% scarlet red ointment or 0.5% silver nitrate solution dressings. Preservation of a healthy excised wound bed and maintenance of a low bacterial count in contaminated wounds paralleled the experience with human allograft dressings despite technical difficulties and the absence of vascularization of amniotic membrane and its fragile structure. Tentative conclusions are drawn as to the mechanisms by which biologic dressings exert their beneficial effects.     

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.     

Dominant Effects of Suppressor of Hairy-Wing Mutations on Gypsy-Induced Alleles of Forked and Cut in Drosophila Melanogaster

Mutations induced by the gypsy retrotransposon in the forked (f) and cut (ct) loci render their expression under the control of the suppressor of Hairy-wing [su(Hw)] gene. This action is usually recessive, but su(Hw) acts as a dominant on the alleles fk, ctk and ctMRpN30. Molecular analysis of the gypsy element present in fk indicates that this allele is caused by the insertion of a modified gypsy in which the region normally containing twelve copies of the octamer-like repeat that interacts with the su(Hw) product is altered. Analysis of the gypsy element responsible for the ctk and ctMRpN30 mutations also reveals a correlation between the dominant action of su(Hw) and disruption of the octamer region. We propose that these disruptions alter the affinity and interaction of su(Hw) protein with gypsy DNA, thereby sensitizing the mutant phenotype to fluctuations in su(Hw) product.     

Inhibition of the Na,K-ATPase by fluoride. Parallels with its inhibition of the sarcoplasmic reticulum CaATPase.

Addition of lithium fluoride to a suspension of Na,K-ATPase undergoing turnover produced a slow (minutes) complete loss of ouabain-sensitive ATPase activity. Persistence of the effect in the presence of deferoxamine showed that fluoride inhibits independent of aluminum. The time course of onset of inhibition was adequately fit by a function corresponding to a monophasic transformation with a pseudo first-order rate constant (k(obs)). This constant varied hyperbolically with [Mg2+] (half-maximal effect at 9 mM Mg2+), whereas it increased with no sign of approaching saturation as the square of [F-], implying that inhibition requires binding of two fluorides/ATPase. The value of k(obs) was found to be increased by greater than 10-fold in the presence of potassium ([K+]1/2 = 0.6 mM) or ouabain. Sodium, ATP, and ADP, which favor the E1 form of the enzyme, had a protective effect. These results implicate the potassium-occluded MgE2(K2) complex as the main fluoride-susceptible form. Protection by Pi and orthovanadate suggests that fluoride exerts its effect at the phosphorylation site. Inhibition was reversible, although slowly, with t1/2 = 7 h at 37 degrees C. Sodium greatly accelerated reversal (t1/2 = 3 min with 150 mM Na+ present), and potassium antagonized this acceleration. The value of k(obs) for reactivation increased steeply with [Na+], with the sodium dependence being about the same at pH 8.0 as at pH 7.4. All of these effects have parallels to effects of fluoride on the sarcoplasmic reticulum CaATPase (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235).     

Studies on isolation and characterization of starch from oat (Avena nuda) grains

Starch from AC Hill oat grains (Avena nuda) was isolated and some of the characteristics determined. The yield of starch was 23·4% on a whole grain basis. The shape of the granule was polyhedral to irregular, with granules 6–10 μm in diameter. Lipids were extracted by acid hydrolysis and by selective solvent extraction with chloroform-methanol 2:1 v/v (CM) at ambient temperature, followed by n-propanol-water 3:1 v/v (PW) at 90–100°C. The acid hydrolyzed extracts which represented the total starch lipids (TSL) was 1·13%. The free lipids in the CM extract (1% TSL) was 6·2%, whereas the free and bound lipids in the PW extracts was 93.0%. Neutral lipids formed the major lipid class in the CM and PW extracts. The monoacyl lipid content in both CM and PW extracts was 61·0%. The total amylose content was 19·4%, of which 13·9% was complexed by native lipids. X-ray diffraction was of the ‘A’ type. Oat starch differed from wheat starch in showing a higher swelling factor, decreased amylose leaching, coleaching of a branched starch component and amylose during the pasting process, higher peak viscosity and set-back, low gel rigidity, greater susceptibility towards acid hydrolysis, greater resistance to -amylase action and a higher freeze-thaw stability. Furthermore, in comparison to wheat starch, the amylose chains of oat starch appear to be more loosely arranged in the amorphous regions, whereas in crystalline regions, oat starch chains are more compactly packed. Lipid removal from oat and wheat starches decreased their swelling factor, peak viscosity, set-back, gelatinization temperatures, freeze-thaw stability and paste clarity (at pH > 4·0), and increased their thermal stability, amylose leaching, enthalpy of gelatinization, susceptibility towards -amylase and paste clarity (at pH < 4·0). The results also showed that the properties of AC Hill oat starch is not representative of oat starch in general.