Miniculture Plaquing Technique for Mouse Cytomegalovirus

A method for plaquing mouse cytomegalovirus under agar in miniature cultures is described. Maximal virus adsorption was found to take approximately 5 hr.     

High levels of RAE-1 isoforms on mouse tumor cell lines assessed by anti-"pan" RAE-1 antibody confer tumor susceptibility to NK cells.

Two sublines of the benzpyrene-induced mouse hepatoma cell line, G-1 and G-5, showed low and high metastatic ability, respectively, to the lung. We produced a polyclonal antibody (pAb) against RAE-1alpha. Five isoforms of RAE-1 have been identified to date, and this pAb recognized all isoforms and was named anti-"pan" RAE-1 pAb. The level of RAE-1 was approximately 5-fold higher in G-5 than in G-1, which was almost RAE-1-negative, as determined using anti-pan RAE-1 pAb. Expression levels of other markers including MHC class I (MHC-I) and Qa-1b were very low and indistinguishable in these sublines. NK-mediated cytotoxicity was determined with these sublines; G-5 was highly susceptible to NK-mediated cytolysis, while G-1 was relatively resistant. The NK-mediated G-5 > G-1 killing profile was diminished if the G-5 cells were pretreated with F(ab)(2)(') of anti-pan RAE-1 pAb. G-1, when transfected with Rae-1alpha cDNA, acquired NK-responsiveness similar to that of G-5. These and additional data using mouse cell lines with low MHC-I levels and various RAE-1 levels also demonstrated that RAE-1 level is critically associated with NK-susceptibility in tumor cells.     

Selective Assembly of V-ATPase Subunit Isoforms in Mouse Kidney

The kidney plays vital roles in acid–base homeostasis, and the reabsorption of water, ions, and proteins. These processes are achieved through acidification of urine and endosomes of proximal tubule epithelial cells. Multisubunit vacuolar-type proton ATPase (V-ATPase) is one of the major acidification-machinery proteins that localizes to the apical or basolateral plasma membranes of intercalated cells in collecting ducts and the endosomal region at the base of brush border microvilli in proximal tubules. Multiple subunit isoforms of V-ATPase, which are expressed in kidney, have been identified. One obvious question is whether the pumps at different locations in the kidney have their own unique subunit identities. We have used a combination of methods to study this enzyme in kidney including immunocytochemical staining and immunoprecipitation analyses. The subunit isoforms of V-ATPase exhibited selective association/assembly in kidney: kidney-specific isoforms predominantly formed the intercalated cell proton pump, whereas the pump located in the brush border comprised ubiquitously expressed counterparts.     

The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity

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Differential Modulation of SERCA2 Isoforms by Calreticulin

In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca²⁺ oscillations in a manner consistent with inhibition of Ca²⁺ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA)2 gene display differential Ca²⁺ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.     

Differential Phosphorylation of Myelin-Associated Glycoprotein Isoforms in Cell Culture

The alternative splicing of myelin-associated glycoprotein (MAG) mRNA generates two isoforms that harbor distinct potential phosphorylation sites in their cytoplasmic tails. Here we characterize the in vivo phosphorylation of MAG isoforms in NIH 3T3 cells transfected with the cDNAs encoding the two isoforms of MAG. Our results demonstrate that the longer isoform, L-MAG, is phosphorylated constitutively mainly on serine, but also on threonine and tyrosine residues. This phosphorylation is subject to change by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ammonium vanadate, but not by dibutyryl-cyclic AMP. The shorter isoform, S-MAG, is constitutively phosphorylated only on serine residues. While TPA and dibutyryl-cyclic AMP have no detectable effect, ammonium vanadate induces tyrosine and threonine phosphorylation in S-MAG. 32P labeling of v-src-transformed NIH 3T3 cells that express L-MAG also show that L-MAG is likely to be an in vivo substrate for pp60v-src tyrosine kinase activity. These results demonstrate that both MAG isoforms are phosphorylated in a heterologous cell system and that this phosphorylation is subject to pharmacological manipulation.     

Tau Isoforms Expression in Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Tau is a protein involved in regulation of microtubule stability, axonal differentiation and transport. Alteration of retrograde transport may lead to motor neuron degeneration. Thus alternative mRNA splicing and expression of tau isoforms were studied in a transgenic mouse model harboring the human SOD1 G93A mutation. The studies were performed on cortex, hippocampus and spinal cord of 64- and 120-day-old animals (presymptomatic and symptomatic stage) and wild type controls. Exon 10 was found in all studied tissues. The 2N isoform containing exons 2 and 3 (+2+3) and the 1N (+2−3) predominated over the 0N (−2−3) in brain regions of the studied mice. The 2N expression was significantly lower in cortex and hippocampus of symptomatic animals compared to analogue control tissues. The decrease in 2N expression resulted in lower levels of total tau mRNA and tau protein. No changes in tau expression were observed in spinal cord of studied animals.     

Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms

Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (α_sk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated α_sk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH₂ terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γ_cyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γ_cyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γ_cyto-actin (but not α_sk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-α_sk-actin or TM-β/γ_cyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γ_cyto-actin (but not α_sk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.     

Novel Isoforms of Mouse Myelin Basic Protein Predominantly Expressed in Embryonic Stage

Abstract: Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well-known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.     

The Susceptibility of Primary Dermis Fibroblasts from the Chinese Tree Shrew to Human Cytomegalovirus Infection

As a universal pathogen leading to neonatal defects and transplant failure, human cytomegalovirus(HCMV) has strict species specificity and this has prevented the development of a suitable animal model for the pathogenesis study. The mechanism of cross-species barrier remains elusive and there are so far no non-human cell culture models that support HCMV replication. The Chinese tree shrew(Tupaia belangeri chinensis) is a small laboratory animal and evolutionary closely related with primates. We investigated the susceptibility of primary tree shrew dermis fibroblasts(TSDF) to HCMV infection. Infection with a GFP-expressing HCMV virus resulted in green fluorescence in infected cells with the expression of IE1, UL44 and pp28. The titers of cell-free viruses reached 103 PFU/mL at 96 hpi, compared to titers of 104 PFU/mL observed in primary human foreskin fibroblasts. Our results suggested that TSDF was semi-permissive for HCMV infection. The TSDF model could be further used to investigate key factors influencing cross-species multiplication of HCMV.     

Metabolic Characterization of a Mouse Deficient in All Known Leptin Receptor Isoforms

We have characterized a newly generated mouse model of obesity, a mouse strain deficient in all five previously described leptin receptor isoforms. These transgenic mice, named the db ₃₃₃/db ₃₃₃ mice, were identified from an ENU mutagenesis screen and carry a point mutation in the seventh exon of the db gene encoding the leptin receptor, resulting in a premature stop codon (Y₃₃₃Stop) and gene product that lacks STAT signaling domains. db ₃₃₃/db ₃₃₃ mice have a morbidly obese phenotype, with body weights diverging from wild type as early as 4 weeks of age (P < 0.05). To determine the contribution of the short isoforms of the leptin receptor in this metabolic phenotype, we performed an extensive metabolic characterization of the db ₃₃₃/db ₃₃₃ mouse in relation to the well-characterized db/db mouse lacking only the long form of the leptin receptor. db ₃₃₃/db ₃₃₃ mice have similar endocrine and metabolic parameters as previously described in other leptin receptor transgenic mice including db/db mice that lack only the long isoform of the leptin receptor. However, db ₃₃₃/db ₃₃₃ mice show a subtle trend toward higher body weight and insulin levels, lower oxygen, carbon dioxide production, respiratory exchange ratio (RER), and temperature than db/db mice suggesting the short isoforms may play an additional role in energy homeostasis.     

Contribution of the Argonaute-1 Isoforms to Invertebrate Antiviral Defense

Argonaute (Ago) protein, the central component of the RNA interference (RNAi) pathway, plays important roles in host innate antiviral immunity. Most organisms harbor a large number of different Ago proteins and isoforms; however, the roles of Ago isoforms in immune defense against pathogens remain unclear. In the present study, three Argonaute-1 (Ago1) isoforms, termed Ago1A, Ago1B, and Ago1C, were found in Marsupenaeus japonicus shrimp. Quantitative real-time PCR (polymerase chain reaction) revealed that isoforms Ago1A and Ago1B containing an insertion sequence in the PIWI domain, were significantly up-regulated in lymphoid organ and hemolymph, and also upon white spot syndrome virus (WSSV) challenge, indicating the involvement of Ago1A and Ago1B in antiviral immunity. The results showed that silencing of Ago1A with a sequence-specific siRNA led to a significant increase of WSSV loads. It was revealed that knockdown of Ago1B mRNA by 37–70% resulted in higher virus loads in shrimp. However, upon silencing Ago1B by more than 85%, a two-fold increase in Ago1A mRNA was observed but viral load was the same as untreated controls challenged with WSSV, suggesting that the simultaneous up-regulation of Ago1A might compensate for the loss of Ago1B. These data indicated that Ago1A played more important roles in the antiviral immune response than Ago1B. The simultaneous inhibition of Ago1A and Ago1B resulted in a greater increase in viral loads than Ago1A or Ago1B alone, indicating that Ago1A and Ago1B isoforms were involved in shrimp antiviral immunity. It was revealed that Ago1C had no effect on virus infection. Therefore, the current study presented the first report on the contribution of Ago isoforms in the invertebrate defense against virus infection.     

Mapping the Genes for Susceptibility and Response to Leishmania tropica in Mouse

Background

L. tropica can cause both cutaneous and visceral leishmaniasis in humans. Although the L. tropica-induced cutaneous disease has been long known, its potential to visceralize in humans was recognized only recently. As nothing is known about the genetics of host responses to this infection and their clinical impact, we developed an informative animal model. We described previously that the recombinant congenic strain CcS-16 carrying 12.5% genes from the resistant parental strain STS/A and 87.5% genes from the susceptible strain BALB/c is more susceptible to L. tropica than BALB/c. We used these strains to map and functionally characterize the gene-loci regulating the immune responses and pathology.

Methods

We analyzed genetics of response to L. tropica in infected F₂ hybrids between BALB/c×CcS-16. CcS-16 strain carries STS-derived segments on nine chromosomes. We genotyped these segments in the F₂ hybrid mice and tested their linkage with pathological changes and systemic immune responses.

Principal Findings

We mapped 8 Ltr (Leishmania tropica response) loci. Four loci (Ltr2, Ltr3, Ltr6 and Ltr8) exhibit independent responses to L. tropica, while Ltr1, Ltr4, Ltr5 and Ltr7 were detected only in gene-gene interactions with other Ltr loci. Ltr3 exhibits the recently discovered phenomenon of transgenerational parental effect on parasite numbers in spleen. The most precise mapping (4.07 Mb) was achieved for Ltr1 (chr.2), which controls parasite numbers in lymph nodes. Five Ltr loci co-localize with loci controlling susceptibility to L. major, three are likely L. tropica specific. Individual Ltr loci affect different subsets of responses, exhibit organ specific effects and a separate control of parasite load and organ pathology.

Conclusion

We present the first identification of genetic loci controlling susceptibility to L. tropica. The different combinations of alleles controlling various symptoms of the disease likely co-determine different manifestations of disease induced by the same pathogen in individual mice.