Granzymes and caspase 3 play important roles in control of gammaherpesvirus latency

Gammaherpesviruses can establish lifelong latent infections in lymphoid cells of their hosts despite active antiviral immunity. Identification of the immune mechanisms which regulate gammaherpesvirus latent infection is therefore essential for understanding how gammaherpesviruses persist for the lifetime of their host. Recently, an individual with chronic active Epstein-Barr virus infection was found to have mutations in perforin, and studies using murine gammaherpesvirus 68 (gammaHV68) as a small-animal model for gammaherpesvirus infection have similarly revealed a critical role for perforin in regulating latent infection. These results suggest involvement of the perforin/granzyme granule exocytosis pathway in immune regulation of gammaherpesvirus latent infection. In this study, we examined gammaHV68 infection of knockout mice to identify specific molecules within the perforin/granzyme pathway which are essential for regulating gammaherpesvirus latent infection. We show that granzymes A and B and the granzyme B substrate, caspase 3, are important for regulating gammaHV68 latent infection. Interestingly, we show for the first time that orphan granzymes encoded in the granzyme B gene cluster are also critical for regulating viral infection. The requirement for specific granzymes differs for early versus late forms of latent infection. These data indicate that different granzymes play important and distinct roles in regulating latent gammaherpesvirus infection.     

Translation elongation factor eEF1A2 is essential for post-weaning survival in mice

Translation elongation factor eEF1A, formerly known as EF-1 alpha, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.     

Alginate-chitosan coacervation in production of artificial seeds

Survival of secondary embryoids of winter oilseed rape (Brassica napus ssp. oleifera cv. Primor) has been used as an assay for the development of artificial seeds involving complex coacervation of alginate (polyanion) with chitosan (polycation). Germination frequency of 100% was achieved for encapsulated embryoids when alginate formed the inner matrix and chitosan the outer layer. When the matrix makeup was reversed, there was no germination of embryoids. The artificial seeds produced were hardened in dilute alkaline solutions of NaOH and Ca(OH)(2). An optimum setting time could be selected based on a quantitative measurement of resistance of hardened capsules to compression and the germination frequency of the encapsulated embryoids. (c) 1993 John Wiley & Sons, Inc.     

Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases

DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity.     

Neurite Outgrowth is Dependent on the Association of c-Src and Lipid Rafts

Regulation of neurite outgrowth is an important aspect not only for proper development of the nervous system but also for tissue regeneration after nerve injury and the treatment of neuropathological conditions. Here, we report that neurite outgrowth in cortical neuron and neuro 2A (N2A) cell was dependent on intact lipid rafts, as well as the enhanced localization of c-Src in the lipid rafts. Src inhibition or lipid rafts disruption could specifically block c-Src phosphorylation profile, pY416 Src increase and pY529 Src decrease, they also resulted in pY529 Src and c-terminal Src kinase (Csk) partition out of lipid rafts. Thus, we concluded that c-Src signal cascades within the lipid rafts is crucial for efficient neurite outgrowth.     

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable f ibronectin‐binding, c ollagen‐binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT‐6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain‐of‐function mutants we show that, unlike other GAS pili, the FCT‐6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT‐6 pili.     

Feedback regulation of the Bradyrhizobium japonicum nodulation genes

Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen-fixing symbiosis with a legume host. The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant-produced flavonoid compounds. The addition of chitin and lipo-chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod–lacZ fusion. Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression. In contrast, expression of the ChiB chitinase increased nod gene expression. The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression. Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2.