Insulin Degrading Enzyme Induces a Conformational Change in Varicella-Zoster Virus gE,and Enhances Virus Infectivity and Stability

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines.     

GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background

Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Methodology/Principal Findings

We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1^−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.

Conclusions/Significance

Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.     

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V_max = 2.31 pmol min^?1 U^?1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.     

Establishment and characterization of a cell line from embryos of the Indianmeal moth,Plodia interpunctella

A cell line, UMN-PIE-1181, initiated in November, 1981, from embryos of a malathion-resistant strain of Indianmeal moth, Plodia interpunctella, was in the 83rd passage on January 28, 1985. The line consists of single, small, fibroblastlike cells that are polyploid with chromosome numbers ranging from 56 to 180. Growth rate is dependent on seeding density, there being no growth at or below seeding densities of $\text{2 ×}\text{10}{}^5\text{5}\text{,}\text{ml}$; optimum growth requires a fetal bovine serum concentration of at least 5%. Twenty-nine isozymes were examined. Five enzymes from the cell lines resolved well and subsequently were compared to enzymes extracted from 4-day-old embryos and other life stages of the insects. Phosphomannose isomerase, malic enzyme, malate dehydrogenase, phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase in extracts from the cultured cells and from the insects had identical patterns. Two bands for glutamate-oxalacetate transaminase, present in the cell line, were not observed in the tissue extracts. Furthermore, lactate dehydrogenase from the cultured cells appeared as four bands but was not detectable in any of the samples run from the various life stages of the insects.     

Modeling acquired immunity as an outcome of the interaction between host-related factors and potential antigen repertoires.

In an attempt to understand why different organisms defend against potential antigens differently, the influence of possible interactions between host-related factors and respective antigen repertoires on the complexity of host defense mechanisms was investigated. A compartmental model coupling these two variables was developed and tested. Data analysis suggests that the more complex the organism, the larger the size of its antigen repertoire. The two variables seem to advance in a parallel fashion suggesting that they could reach a state of equilibrium. Therefore, host-related factors may play a role in determining the size of the antigen repertoire on the one hand; on the other hand, increased antigen repertoire size may dictate the evolution of more complex mechanisms of immunity. Although the interplay between the two variables maintains some common themes in different groups of organisms, it results in clear differences pertinent to immunologic specificity, diversity, memory and self nonself discrimination.     

Ovarian dynamics and milk progesterone concentrations in cycling and non-cycling buffalo-cows (Bubalus bubalis) during Ovsynch program

The objective was to evaluate ovarian dynamics and progesterone concentrations in cyclic (CYC, n=10) and non-cyclic (NCY, n=8) buffalo-cows during Ovsynch program. All cows received GnRH on day 0, PGF2alpha on day 7, and GnRH on day 9, and AI 14 h later. Ovarian structures were monitored by ultrasound and milk samples were collected for progesterone (P4) analysis. The first GnRH resulted in ovulation in CYC (90%) and NCY (62.5%) cows. By day 7, almost all cows had large follicle and lutein tissue. Luteolytic responses to PGF2alpha were 80 and 87.5% for CYC and NCY cows, respectively. Following second GnRH, ovulation occurred in 80% of CYC and 100% of NCY cows. Ovulation began earlier (12 h following second GnRH) and extended for longer (36 h) in NCY cows, when compared to CYC cows (36 and 12 h, respectively). The mean P4 levels increased from days 0 through 7 in CYC and NCY cows and levels were higher in CYC group. Conception rates were 60 and 37.5% in CYC and NYC cows, respectively. Early and asynchronous ovulation and luteal sub-function seemed to be a problem in NCY cows. Inseminating NCY cows twice, at 0 and 24 h of the second GnRH is recommended.     

Molecular characterization of Cryptosporidium isolates from humans and animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

Alginate as an antiglycating agent for human serum albumin

Hyperglycemia and the accumulation of advanced glycation endproducts (AGEs) in tissues and serum have important roles in diabetic complications. Therefore, the identification of anti-glycation compounds is attracting considerable interest. In this study, the interaction of human serum albumin (HSA) with fructose, in the absence and presence of alginate, was studied by circular dichroism, absorbance and fluorescence techniques. The characterization study of AGEs was performed using autofluorescence, fibrillar formation, the increase in absorbance and the quantification of free lysine side chains. The results indicate that alginate inhibits the fructation of HSA as observed by a reduction in the formation of fluorescent AGEs and fibrils. Furthermore, alginate reduces the amount of modified lysine side chains, signified by the lack of increase in absorbance, and increases the helicity of this protein.     

Radical disinfestation protocol eliminates in vitro contamination in Guava (<Emphasis Type="Italic">Psidium guajava </Emphasis>L.) seeds

Controlling contamination in vitro is one of the basic requirements for successful tissue culture technology. In preliminary studies, in vitro contamination of guava seeds was almost 100%, even when disinfested with bleach. To overcome this problem, we developed a unique method to control contamination using bleach and strong acids. Submerging guava seeds in 10% HCl for 24–72 h followed by a 30 min treatment with 10% bleach (NaOCl) reduced contamination rates from 98 to 0%. Substituting 5% H₂SO₄ for 12 h for 10% HCL gave similar results. In addition to eliminating contamination, acid-treated seeds germinated faster in vitro than control seeds germinated in a greenhouse (15 vs. 40 days, respectively).