A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Amine Accumulation in Acidic Vacuoles Protects the Halotolerant Alga Dunaliella salina Against Alkaline Stress

Amines at alkaline pH induce in cells of the halotolerant alga Dunaliella a transient stress that is manifested by a drop in ATP and an increase of cytoplasmic pH. As much as 300 millimolar NH₄⁺ are taken up by the cells at pH 9. The uptake is not associated with gross changes in volume and is accompanied by K⁺ efflux. Most of the amine is not metabolized, and can be released by external acidification. Recovery of the cells from the amine-induced stress occurs within 30 to 60 minutes and is accompanied by massive swelling of vacuoles and by release of the fluorescent dye atebrin from these vacuoles, suggesting that amines are compartmentalized into acidic vacuoles. The time course of ammonia uptake into Dunaliella cells is biphasic—a rapid influx, associated with cytoplasmic alkalinization, followed by a temperature-dependent slow uptake phase, which is correlated with recovery of cellular ATP and cytoplasmic pH. The dependence of amine uptake on external pH indicates that it diffuses into the cells in the free amine form. Studies with lysed cell preparations, in which vacuoles become exposed but retain their capacity to accumulate amines, indicate that the permeability of the vacuolar membrane to amines is much higher than that of the plasma membrane. The results can be retionalized by assuming that the initial amine accumulation, which leads to rapid vacuolar alkalinization, activates metabolic reactions that further increase the capacity of the vacuoles to sequester most of the amine from the cytoplasm. The results indicate that acidic vacuoles in Dunaliella serve as a high-capacity buffering system for amines, and as a safeguard against cytoplasmic alkalinization and uncoupling of photosynthesis.     

Hydrolysis of polyphosphates and permeability changes in response to osmotic shocks in cells of the halotolerant alga dunaliella

The effects of osmotic shocks on polyphosphates and on the vacuolar fluorescent indicator atebrin have been investigated to test whether acidic vacuoles in the halotolerant alga Dunaliella salina have a role in osmoregulation. Upshocks and downshocks induce different patterns of polyphosphate hydrolysis. Upshocks induce rapid formation of new components, tentatively identified as 5 or 6 linear polyphosphates, formed only after upshocks with NaCl and not with glycerol, indicative of compartmentation of Na⁺ into the vacuoles. Conversely, downshocks induce a slower transient accumulation of tripolyphosphates, indicating activation of a different hydrolytic process within the vacuoles. Osmotic shocks do not lead to release of atebrin from acidic vacuoles, indicating that they do not induce a major intravacuolar alkalinization. However, osmotic shocks induce transient permeability changes measured by amine-induced atebrin release from vacuoles. Hypoosmotic shocks transiently increase the permeability (up to 20-fold), whereas hyperosmotic shocks induce a rapid drop in permeability. Electron micrographs of osmotically shocked cells also reveal transient changes in the surface and internal organelles of D. salina cells. It is suggested that hyperosmotic and hypoosmotic shocks induce different changes within acidic vacuoles and in the organization and/or composition of the plasma membrane in Dunaliella.     

Management of the recalcitrant total-hip arthroplasty wound

The infection rate for total-hip arthroplasty is around 1 percent. This small group is usually managed by complete removal of the prosthesis and the cement and closure over suction catheters to "collapse" the wound and eventually achieve a girdlestone arthroplasty. Occasionally, there are patients who have a persistent draining wound after this treatment and repeated efforts at wound closure. We present 27 patients who had recalcitrant, noncollapsible wounds of the hip that were present for many months to years. Twenty-eight cases of infected total-hip arthroplasties that did not respond to removal of the prosthesis and cement and closure were seen by the authors between January of 1977 and December of 1988. One patient had bilateral involvement. Average age was 64 years (range 33 to 79 years). There was an average of 4.2 previous surgical attempts at closure (range 1 to 21). Staphylococcus aureus was the most common organism, but the infections were virtually all multiple. Thirty-three muscles were utilized in 27 patients. The rectus femoris was used in 23 cases, the vastus lateralis in 8, tensor fasciae latae in 1, and combined latissimus dorsi-serratus anterior free-tissue transfers were carried out in 2. Multiple combinations of transpositions and free flaps were utilized. Follow-up ranged from 1 to 10 years, with an average of 6.4 years. Eighteen patients were ambulatory with minor degrees of pain, five ambulated with a cane, seven ambulated with a walker, six ambulated with crutches, and four ambulated unassisted, all of whom had reimplantation of their hip arthroplasty at least 12 months following the muscle flap procedure.(ABSTRACT TRUNCATED AT 250 WORDS)     

A light-dependent dicyclohexylcarbodiimide-sensitive Ca-ATPase activity in chloroplasts which is not coupled to proton translocation

The early observation of light-dependent Ca-ATPase activity in chloroplast thylakoids [Avron, M. (1962) J. Biol. Chem. 237, 2011-2017] has been reinvestigated. It is demonstrated that in contrast to light-triggered Mg-ATP activity, Ca-ATPase activity is strictly dependent on delta microH+, the transthylakoid membrane electrochemical potential gradient, since (a) there is an absolute requirement for continuous illumination; (b) electron-transport mediators that catalyze proton uptake, like phenazinemethosulphate, methylviologen of ferricyanide, are essential and (c) uncouplers inhibit the activity. The Ca-ATPase activity is essentially unaffected by dithiols, but is inhibited by CF0-CF1 inhibitors including tentoxin, dicyclohexylcarbodiimide and antisera to CF1. Addition of Ca-ATP to thylakoids does not induce delta pH or delta psi (the electrical potential gradient) formation either in the light or following preillumination with dithiols, demonstrating that it is not coupled to proton translocation. It is also demonstrated that Ca-ATP or Ca-ADP does not induce a proton leak through CF0-CF1. It is concluded that the Ca-ATPase activity in chloroplast thylakoid reflects a partial reaction of ATP synthesis catalyzed by CF0-CF1, which is internally uncoupled from proton translocation but is dependent on energization by a transmembrane delta microH+.