Species-Specific Allozyme Markers for Appalachian Wood-Feeding Cockroaches (Dictyoptera: Cryptocercidae)

Members of the genus Cryptocercus are wood-feeding cockroaches that live in the temperate forests. Nine species are recognized in the genus worldwide: two in eastern Eurasia, two in China, and five in the United States. Within the United States, one species occurs in the Pacific Northwest and four occur in the Appalachian Mountains. Previous studies have revealed the presence of potential zones of overlap in distribution among the Appalachian species, raising the possibility of hybridization among them. Differences in mitochondrial DNA have previously been identified for the Appalachian species. However, to identify hybrid individuals one or more species-specific, codominant nuclear markers are required. Therefore, our objective was to undertake allozyme analysis of enzymatic loci to identify fixed, species-specific alleles for the four Appalachian species. We assayed a mean of 42 individuals each from 16 sites for allozyme variation for the four species. At 6 of the 33 loci examined, fixed alternate alleles were identified; a combination of 2 loci enabled the identification of all four species. To identify hybrids in the field, we examined 42 individuals each from 13 sites in which two or more of the above species occur in close proximity for presence of heterozygous individuals at one or more of the six fixed loci. No heterozygous individuals were found suggesting the lack of hybridization among the Appalachian species.     

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.     

Cloning, characterization, and functional expression of the Klebsiella oxytoca xylodextrin utilization operon (xynTB) in Escherichia coli

Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na(+)/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.     

Influence of conservation on calculations of amino acid covariance in multiple sequence alignments

It has long been argued that algorithms that find correlated mutations in multiple sequence alignments can be used to find structurally or functionally important residues in proteins. We examined the properties of four different methods for detecting these correlated mutations. On both simple, artificial alignments and real alignments from the Pfam database, we found a surprising lack of agreement between the four correlated mutation methods. We argue that these differences are caused in part by differing sensitivities to background conservation. Correlated mutation algorithms can be envisioned as "filters" of background conservation with each algorithm searching for correlated mutations that occur at a different background conservation frequency.     

Effects of intestinal survival surgery on systemic and mucosal immune responses in SIV-infected rhesus macaques

Abstract: Evaluation of cellular immunity in the intestinal lamina propria of rhesus macaques has been used previously to assess protective immunity against mucosal simian immunodeficiency virus (SIV) challenges. As this technique requires survival surgery to obtain jejunal tissue, effects of surgical stress on the immune system were investigated. SIV-specific immune responses, including IgG and IgA binding antibodies in sera and mucosal secretions, IgG and IgA secreting cells in peripheral blood, IgG neutralizing antibodies, T-cell proliferative responses, and interferon-γ secretion by peripheral blood mononuclear cells, were evaluated pre- and post-surgery in macaques immunized with adenovirus-SIV recombinant vaccines and SIV envelope protein and in SIV-infected macaques. No differences in these immune parameters were observed in SIV-naïve, immunized macaques or healthy SIV-infected macaques with regard to surgery. A dramatic increase in total IgA antibody level following surgery in the rectal secretions of one SIV-infected macaque that was rapidly progressing to AIDS and failed to recover from surgery was attributed to an abscess that developed at the intestinal site. To date, nearly 30 other macaques have undergone the intestinal survival surgery, some on more than one occasion, without experiencing any clinical difficulty. Overall, our results suggest that in healthy macaques, intestinal resection survival surgery can be conducted safely. Further, the method can be used to reliably sample the intestinal mucosa without major or persistent impact on humoral or cellular immune responses.     

Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon Methanosarcina thermophila

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α₇ ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr¹ residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys³³ with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys³³ in polarization of the Thr¹ N, which serves to strip a proton from the active-site Thr¹ Oγ nucleophile. Replacement of Asp⁵¹ with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.     

Suppression of Colorado potato beetle infestation by pheromone-mediated augmentation of the predatory spined soldier bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae)

1 Hardware and protocols were tested to enable individual growers and insectary operators to mass-produce predatory spined soldier bugs (SSBs), Podisus maculiventris (Say) (Heteroptera: Pentatomidae: Asopinae), for augmentative biological control. Using pheromone-based technology, an average of 1775 female SSBs (potentially as many as 1.6 million offspring) were captured each year during 2–3 weeks in early spring. 2 Data for the first 2 years of a 3-year project to use SSB for biological suppression of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), verified earlier research showing that augmentation of SSB (～5 nymphs/plant) can significantly suppress CPB infestations. In the third year, wild SSBs were transferred directly from pheromone traps to mid-plot nursery cages having a mesh size chosen to retain the adult predators but allow their offspring to escape. Pheromone dispensers were placed peripherally to promote dispersal of young predators and immigration of new wild spined soldier bug adults. Pheromone-mediated augmentation using porous nursery cages and pheromone dispensers was less labour-intensive than earlier methods, and resulted in significantly improved potato yield. 3 Trapping SSB adults early in the spring protects them from parasitization by tachinid flies and scelionid wasps that use the pheromone to facilitate host-finding. The compatibility of pheromone-mediated predator augmentation/conservation with implementation of transgenic plants, imidacloprid insecticide, and other biocontrol methods is discussed.     

Transfer of Methanolobus siciliae to the genus Methanosarcina, naming it Methanosarcina siciliae, and emendation of the genus Methanosarcina

A sequence analysis of the 16S rRNA of Methanolobus siciliae T4/M(T) (T = type strain) showed that this strain is closely related to members of the genus Methanosarcina, especially Methanosarcina acetivorans C2A(T). Methanolobus siciliae T4/M(T) and HI350 were morphologically more similar to members of the genus Methanosarcina than to members of the genus Methanolobus in that they both formed massive cell aggregates with pseudosarcinae. Thus, we propose that Methanolobus siciliae should be transferred to the genus Methanosarcina as Methanosarcina siciliae.     

Ultrastructural effects of Helminthosporium maydis race T toxin on mitochondria of corn roots and protoplasts

Zea mays inbred W64A in Texas (T, toxin sensitive) male sterile and non-male sterile (N, toxin resistant) cytoplasms were utilized. Roots of freshly germinated seeds were treated for 15 min of 2 hr with culture filtrate from liquid grown Helminthosporium maydis Race T, or with a chloroform extractable purified fraction from the culture filtrate. In the susceptible W64A T line, toxin treatment, both crude and purified, caused swelling and loss of matrix densiy in mitochondria of root cap and vacuolated cells in the region of elongation. One hour treatment with the chloroform extractable toxin fraction caused similar effects or mitochondria of isolated leaf protoplasts. This is the first report of such rapid in vivo effects of HmT toxin on mitochondria. Difficulty in obtaining consistent preservation of meristem mitochondria precluded drawing firm conclusions concerning that region of the root. In the resistant W64A N line, protoplast and root mitochondria were unaffected by the toxin.     

The flax ribosomal RNA-encoding genes are arranged in tandem at a single locus interspersed by ‘non-rDNA’ sequences

The ribosomal RNA (rRNA)-encoding genes (rDNA) in flax, estimated to be present in about 2400 copies per diploid nucleus, have been reported as a single homogeneous repeat unit of 8.6 kb. In situ hybridization analysis indicated that these genes were located at a single site on one pair of chromosomes. However, an analysis of a flax variety, CI 1303, has revealed heterogeneity in the intergenic spacer of the rDNA repeat unit. A genetic analysis of rDNA inheritance in two flax lines, Stormont Cirrus and CI 1303, has again supported the observation that there is a single rDNA locus in this plant species. Screening of four different genomic libraries made in methylation-sensitive and -insensitive systems, and the analysis of 40 phage clones, demonstrate a much higher number than that expected of junctions between rDNA and non-rDNA. Direct evidence of rRNA-encoding genes being present in tandem comes from a few phage clones that contain more than two rDNA repeats. The evidence presented here indicates that rDNA, although present at a single locus in tandem arrays, may be interrupted frequently by other non-rDNA sequences, thus giving rise to questions about their organization into long tandem arrays.