Identification of an essential virulence gene of cyprinid herpesvirus 3

The genus Cyprinivirus consists of a growing list of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. The archetypal member, cyprinid herpesvirus 3 (CyHV-3) causes mass mortalities worldwide in koi and common carp. A CyHV-3 mutant was described previously that is attenuated in vivo by a deletion affecting two genes (ORF56 and ORF57). The relative contributions of ORF56 and ORF57 to the safety and efficacy profile of this vaccine candidate have now been assessed by analysing viruses individually deleted for ORF56 or ORF57. Inoculation of these viruses into carp demonstrated that the absence of ORF56 did not affect virulence, whereas the absence of ORF57 led to an attenuation comparable to, though slightly less than, that of the doubly deleted virus. To demonstrate further the role of ORF57 as a key virulence factor, a mutant retaining the ORF57 region but unable to express the ORF57 protein was produced by inserting multiple in-frame stop codons into the coding region. Analysis of this virus in vivo revealed a safety and efficacy profile comparable to that of the doubly deleted virus. These findings show that ORF57 encodes an essential CyHV-3 virulence factor. They also indicate that ORF57 orthologues in other cypriniviruses may offer promising targets for the rational design of attenuated recombinant vaccines

A role for calcineurin in Dictyostelium discoideum phagocytosis

The Ca2+/calmodul1n-dependent protein phosphatase calcinewin is involved in the development of the cellular slime mold Dictyostelium discoideum. Because of its interactions with Ca2+, which appear to influence D. discoideum phagocytosis (Yuan and Chia, 1999, Mol. Biol. Cell 10, 220a), we undertook studies to test whether calcineurin also plays a role in Dictyostelium phagocytosis. The immunosuppressants cyclosporin A and FK506, through the formation of cyclosporin A-cyclophilin A and FK506- FK506-binding protein complexes, respectively, inhibited calcineurin activity. These two calcineurin inhibitors suppressed phagocytosis of fluorescently labeled yeast in a dose-dependent manner. Although it inhib~ted phagocytosis, cyclosporin A had an insignificant effect on the macropinocytosis of the fluid-phase marker fluorescein isothiocyanatedextran. Furthermore, trifluoperazine, a calmodulin antagonist that indirectly inhibits calcinewin, also suppressed phagocytosis in a dosedependent fashion and induced the formation of giant intracellular vacuoles Fluorescence microscopy of cyclosporin A-treated (for 30 min.) cells stained with rhodamine-phalloidin had cytoplasmic chunks of F-actin that were not present in control cells, while cells treated with FK506 and trifluoperazine (also for 30 min.), displayed less cortical but more cytoplasmic F-actin staining than normal cells. Typically, drug-treated cells were smaller and rounder than untreated cells. Our data suggest calcineurin may play a role in D. discoideum phagocytosis, either through the dephosphorylation of actinregulating proteins or other cytoskeletal proteins such as the heavy chain subunit of nonmuscle myosin I1 since dephosphorylation of the latter promotes filament assembly

A role for calcineurin in Dictyostelium discoideum phagocytosis

The Ca2+/calmodul1n-dependent protein phosphatase calcinewin is involved in the development of the cellular slime mold Dictyostelium discoideum. Because of its interactions with Ca2+, which appear to influence D. discoideum phagocytosis (Yuan and Chia, 1999, Mol. Biol. Cell 10, 220a), we undertook studies to test whether calcineurin also plays a role in Dictyostelium phagocytosis. The immunosuppressants cyclosporin A and FK506, through the formation of cyclosporin A-cyclophilin A and FK506- FK506-binding protein complexes, respectively, inhibited calcineurin activity. These two calcineurin inhibitors suppressed phagocytosis of fluorescently labeled yeast in a dose-dependent manner. Although it inhib~ted phagocytosis, cyclosporin A had an insignificant effect on the macropinocytosis of the fluid-phase marker fluorescein isothiocyanatedextran. Furthermore, trifluoperazine, a calmodulin antagonist that indirectly inhibits calcinewin, also suppressed phagocytosis in a dosedependent fashion and induced the formation of giant intracellular vacuoles Fluorescence microscopy of cyclosporin A-treated (for 30 min.) cells stained with rhodamine-phalloidin had cytoplasmic chunks of F-actin that were not present in control cells, while cells treated with FK506 and trifluoperazine (also for 30 min.), displayed less cortical but more cytoplasmic F-actin staining than normal cells. Typically, drug-treated cells were smaller and rounder than untreated cells. Our data suggest calcineurin may play a role in D. discoideum phagocytosis, either through the dephosphorylation of actinregulating proteins or other cytoskeletal proteins such as the heavy chain subunit of nonmuscle myosin I1 since dephosphorylation of the latter promotes filament assembly

Integrity of the actin cytoskeleton required for both phagocytosis and macropinocytosis in \u3ci\u3eDictyostelium discoideum\u3c/i\u3e

Filamentous (F-) actin is enriched in cellular extensions, such as phagocytic cups and macropioocytic crowns, of Dlctyostelium discoideum amebae. Previous studies of actin-disrupting agents that implicated the involvement of the actin cytoskeleton in Dictyostelium phagocytosis and pinocytosis, however, have yielded conflicting results. We show that the integrity of the actin cytoskeleton is required for both phagocytosis and macropinocytosis in D. discoideum with latrunculin A (IatA), which binds to monomeric actin, and cytochalasin A (cytA), which caps the plus end of actin filaments. Using rhodamine-phalloidin to visualize F-actin, cells treated for 30 min. with 1 to 4 pM of latA displayed an increasing dissolution of the cortical actin cytoskeleton that was accompanied by the appearance of numerous cytoplasmic dots of F-actin. In parallel, phagocytosis of fluorescently labeled yeast and macropinocytosis of the fluid-phase marker fluorescein isothiocyanate-dextran both were inhibited in a dose-dependent manner. Cells were nearly devoid of F-actin at latA concentrations greater than 5pM whereas the uniform distribution of monomeric actin appeared unaffected. Cells gradually recovered their intact actin cytoskeleton and concomitantly, their phagocytic and macropinocytic activities when latA was removed by washing. To achieve 50% inhibition of phagocytosis or macropinocytosis, five-fold more cytA than latA was required. Unlike latA-treated cells, cytAtreated cells stained with rhodamine phalloidin retained an actin cytoskeleton even at high concentrations (\u3e25 μM), but were smaller and rounder than untreated cells. The cortical F-actin, however, appeared irregular, and almost discontinuous, which made the cells seem stiff and rigid in comparison to normal cells that looked more fluid and plastic. The distinctive alterations in the cytoskeletal patterns reflected the specific modes of action of the drugs on the actin network that was vital for both phagocytosis and macropinocytosis

Nonspecific interactions alter lipopolysaccharide patterns and protein mobility on sodium dodecyl sulfate polyacrylamide gels

In testing whether bacterial lipopolysaccharide (LPS) was a natural substrate for an esterase from the soil amebae Dictyostelium discoideum, we observed altered banding patterns of the LPS and changed protein mobility on sodium dodecyl sulfate (SDS) polyacrylamide gels after incubation of LPS with the enzyme. The initial interpretation of these results was that the enzyme had removed ester-linked acyl chains from the LPS, leading to a change in its migration on gels. However, esterase inactivated by treatment with either dithiothreitol (DTT), heat, or SDS generated the same mobility shifts. Bovine serum albumin (BSA) also induced the same change in the electrophoretic pattern. We conclude that the altered LPS patterns and protein mobility on SDS gels were caused by nonspecific interactions between LPS and protein

Nonspecific interactions alter lipopolysaccharide patterns and protein mobility on sodium dodecyl sulfate polyacrylamide gels

In testing whether bacterial lipopolysaccharide (LPS) was a natural substrate for an esterase from the soil amebae Dictyostelium discoideum, we observed altered banding patterns of the LPS and changed protein mobility on sodium dodecyl sulfate (SDS) polyacrylamide gels after incubation of LPS with the enzyme. The initial interpretation of these results was that the enzyme had removed ester-linked acyl chains from the LPS, leading to a change in its migration on gels. However, esterase inactivated by treatment with either dithiothreitol (DTT), heat, or SDS generated the same mobility shifts. Bovine serum albumin (BSA) also induced the same change in the electrophoretic pattern. We conclude that the altered LPS patterns and protein mobility on SDS gels were caused by nonspecific interactions between LPS and protein

Assessing the safety and efficacy of switching to brinzolamide/timolol fixed combination as a replacement therapy in patients with uncontrolled intraocular pressure in Taiwan

AbstractPurposeThe objective of this study is to assess the safety and efficacy of switching to brinzolamide 1% and timolol 0.5% fixed combination (BTFC) from prior pharmacotherapy in patients with open-angle glaucoma (OAG) or ocular hypertension (OH) in Taiwan.MethodsThis was a multicenter, open-labeled, interventional prospective study. The 8-week study involved patients with OAG or OH with uncontrolled intraocular pressure (IOP) and consisted of three study visits to the clinical site. Patients were instructed to discontinue their prior medications at the first visit, prior to starting the study medication. Enrolled patients were dosed with BTFC twice daily in both eyes for 8 weeks. IOP measurements and safety evaluations were conducted at both Week 4 and Week 8.ResultsA total of 74 patients were enrolled. The overall mean IOP reductions from baseline after Week 8 of BTFC was 3.45 mmHg (15.42%); when subgrouped by prior medication class (β-blockers vs. non-β-blockers), the reduction in mean IOP after transitioning to BTFC at Week 8 was as follows: subgroup β-blockers were 3.23 mmHg (14.9 %) and non-β-blockers were 3.58 mmHg (15.25%). All mean IOP changes from baseline were statistically significant (p < 0.001). Of the 69 patients (per protocol population) who were switched to BTFC regardless of prior therapy, 37 (53.6%) patients at Week 4 and 38 (55.1%) patients at Week 8 had IOP ≤ 18 mmHg. No treatment-related serious adverse events were reported in this study.ConclusionThe results of this study demonstrated the potential benefit of using BTFC as a replacement therapy in order to ensure adequate IOP control. BTFC administered twice daily was safe and effective in patients with uncontrolled IOP in Taiwan

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[35S]methionine into the lumbar spinal cord. 2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules. 3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin. 4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[35S]methionine into the lumbar spinal cord. 2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules. 3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin. 4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form