Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Membrane compartmentalized ATP and its preferential use by the Na, K-ATPase of human red cell ghosts

This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na, K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [γ-(32)P]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37 degrees C, but not at 0 degrees C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [gamma-(32)P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as (32)P(i) liberated during the subsequent incubation at 0 degrees C, reflects completely the differential effects of preincubation with ATP on (32)P incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na, K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na, K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.     

Rimantadine but not amantadine protects Fischer rat embryo cells from transformation induced by 3-methylcholanthrene or benzo(a)pyrene.

The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimanbadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene.     

Control of simian/human immunodeficiency virus viremia and disease progression after IL-2-augmented DNA-modified vaccinia virus Ankara nasal vaccination in nonhuman primates

A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-gamma-secreting cells and TNF-alpha-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.     

Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.     

Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.     

Identifying the target cell in primary simian immunodeficiency virus (SIV) infection: highly activated memory CD4(+) T cells are rapidly eliminated in early SIV infection in vivo

It has recently been shown that rapid and profound CD4(+) T-cell depletion occurs almost exclusively within the intestinal tract of simian immunodeficiency virus (SIV)-infected macaques within days of infection. Here we demonstrate (by three- and four-color flow cytometry) that this depletion is specific to a definable subset of CD4(+) T cells, namely, those having both a highly and/or acutely activated (CD69(+) CD38(+) HLA-DR(+)) and memory (CD45RA(-) Leu8(-)) phenotype. Moreover, we demonstrate that this subset of helper T cells is found primarily within the intestinal lamina propria. Viral tropism for this particular cell type (which has been previously suggested by various studies in vitro) could explain why profound CD4(+) T-cell depletion occurs in the intestine and not in peripheral lymphoid tissues in early SIV infection. Furthermore, we demonstrate that an acute loss of this specific subset of activated memory CD4(+) T cells may also be detected in peripheral blood and lymph nodes in early SIV infection. However, since this particular cell type is present in such small numbers in circulation, its loss does not significantly affect total CD4(+) T cell counts. This finding suggests that SIV and, presumably, human immunodeficiency virus specifically infect, replicate in, and eliminate definable subsets of CD4(+) T cells in vivo.     

A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid-potentiated oxidative burst

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild-type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S-SH-L). Challenge of 35S-SH-L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild-type tobacco, both the onset of plant cell death and the induction of an active oxygen species-responsive promoter (AoPR1-GUS) were delayed following challenge of 35S-SH-L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild-type tobacco, whereas in 35S-SH-L plants the onset of H2O2 accumulation was delayed by 2-3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1-2 h following inoculation in wild-type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence-gene induction, factors that together may determine the outcome of plant-pathogen interactions.     

Do enzymatic hydrolyzability and Simons' stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes?

In an attempt to elucidate the impact of substrate accessibility to cellulases on the susceptibility of lignocellulosic substrates to enzymatic hydrolysis, a hydrogen peroxide treated, Douglas fir kraft pulp was dried using several methods with varying levels of intensity. Oven-drying at 50 and 100 degrees C, air-drying, and freeze-drying methods were employed to remove the interfibrillar water from the pulp samples. Subsequently, the never-dried and variably dried pulps were hydrolyzed using a commercial cellulase preparation supplemented with additional beta-glucosidase. Drying reduced the susceptibility of the substrates to enzymatic hydrolysis, which can be attributed to the hornifying effect that drying has on fibers. This effect was more pronounced for the fibers that were oven-dried at 100 degrees C (23% reduction) and 50 degrees C (15% reduction), and there was a good correlation between the Simons's stain results and the enzymatic digestibility of the dried pulps. These observations indicated that drying significantly reduced the population of larger pores and that the partial closure of larger pores created a large number of smaller pores that were not accessible to the displacement dye molecules (orange dye). The inaccessibility of the cellulose to the enzymes, due to the collapse or closure of the large pores, appears to be the primary reason for the lower susceptibility of the dried pulps to enzymatic hydrolysis.     

An outbreak of besnoitiosis in miniature donkeys

Fourteen miniature donkeys (Equus asinus) in a mid-Michigan herd of 38 animals presented with clinical signs of besnoitiosis, including the presence of typical tissue cysts in the ocular sclera, the buccal and nasal mucosa, together with characteristic dermatitis in specific areas of the body. The common histopathological change seen was the presence of many 100-200-microm diameter, thick walled, typical Besnoitia sp. tissue cysts together with a chronic cellular response associated with degenerating cysts. Microscopy of isolated scleral cysts and skin biopsies showed the presence of protozoal organisms consistent in morphology with that of Besnoitia bennetti bradyzoites. Molecular analysis of these parasites indicates that they differ from previously described coccidia, including Besnoitia sp., from rabbits and opossums. Isolated cases of infection with this agent have been reported infrequently in equids; however, this is the first report of an outbreak in a herd of donkeys in the United States.