Poly(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(β-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760–12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(β-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation. Received: 26 May 1999 / Accepted: 16 June 1999     

An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cell mitochondrial DNA

The 4S RNA genes in HeLa mitochondrial DNA (mtDNA) have been mapped by electron microscopy using the electron-opaque label ferritin. This method is based on the high affinity interaction between the protein, avidin, and biotin. 4S RNA, covalently coupled to biotin, was hybridized to single-stranded mtDNA. The hybrids were then labeled with ferritin-avidin conjugates. The positions of ferritin-labeled 4S RNA genes were determined relative to the rRNA genes on both heavy (H) and light (L) strands of mtDNA. This region was recognized as a duplex segment after hybridization either with rRNA in the case of H strands or with DNA complementary to rRNA in the case of L strands.Our studies suggest that at least nineteen 4S RNA genes are present in the HeLa mitochondrial genome. On the H strand, we have confirmed the nine map positions found in a previous electron microscope mapping study (Wu et al., 1972) and obtained evidence for three additional 4S RNA genes. On the L strand, seven 4S RNA genes have been mapped. The nineteen genes are distributed more or less uniformly around the genome. There is a pair of closely spaced genes, approximately 150 nucleotides apart, on the H strand, and another closely spaced pair on the L strand.     

Preadolescents’ relationships with pet dogs: Relationship continuity and associations with adjustment

Research on human–animal interaction in children has been studied in isolation rather than integrated with core theories of children’s relationships. This study is one of the first to examine how children’s relationships with pet dogs are related to their human relationships (parent–child attachments, friendships) and to child adjustment, and to include observational assessment of children’s interactions with their pet dog. Children (9 to 11 years old, n = 99) completed questionnaires regarding relationships with pet dogs, parents, and friends. Half the children were observed interacting with their pet dog. Children and teachers reported children’s adjustment. Children who felt closer to their dogs were more securely attached to mothers and fathers and reported more positive qualities and less conflict with friends. Children with more secure attachments to mothers, and greater companionship with dogs, interacted more with their dogs. Parental attachment and friendship quality, but not the pet dog relationship, were related to child adjustment.     

Proliferation and differentiation of ductular progenitor cells and littoral cells during the regeneration of the rat liver to CCl4/2-AAF injury

Restoration of centrolobular injury induced by carbon tetrachloride (CCl4), when hepatocyte proliferation is inhibited by treatment with N-2-acetylaminofluorene (AAF), is accomplished by proliferation of ductular progenitor cells, that arise intraportally and extend into the liver lobule. This pattern contrasts to the restitutive proliferation of hepatocytes when AAF is not administered, and the proliferation of non-ductular periportal oval cells follows periportal necrosis induced by allyl alcohol. The expanding ducts stain for alphafetoprotein (AFP), OV-6, pan-cytokeratin (CKPan), and laminin. The neoductular proliferation is accompanied by fibronectin-positive Kupffer cells and desmin-positive stellate (Ito) cells, which may play critical roles not only in controlling proliferation and differentiation of ductular progenitor cells, but also in reestablishing hepatic cord structure. When AAF is discontinued 7 days after injury, clusters of small hepatocytes appear next to the neoductules. Some of these small hepatocytes, as well as some larger hepatocytes adjacent to the ducts, stain for AFP and for carbamoylphosphate synthetase I (CPS-I), suggesting that the ductular progenitor cells may differentiate into hepatocytes when AAF is withdrawn. The restitutive process is facilitated by clearing of the central necrotic zone by infiltrating macrophages and co-migration of mature hepatocytes, with Kupffer cells and stellate cells, into the necrotic zone.     

Composite Sampling of a Bacillus anthracis Surrogate with Cellulose Sponge Surface Samplers from a Nonporous Surface

A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm² (four separate 929 cm² areas), larger than the 645 cm² prescribed by the standard Centers for Disease Control (CDC) and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261). Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720) for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001). The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.     

A recombinant multivalent combination vaccine protects against Chlamydia and genital herpes

Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) genital infections pose a considerable public health challenge worldwide. Considering the high incidence of coinfections by the two pathogens, a combination vaccine that can be administered as a single regimen would be highly desirable. Recombinant Vibrio cholerae ghosts (rVCG) offer an attractive approach for the induction of humoral and cellular immune responses against human and animal pathogens. In this study, we evaluated a bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D in mice for immunogenicity and protective efficacy against genital challenge with either pathogen. Mice immunized with the combination vaccine elicited secretory IgA and IgG2a antibodies to both chlamydial and HSV-2 antigens in serum and vaginal secretions. Robust antigen-specific mucosal and systemic T helper type 1 responses were induced in mice as measured by increased interferon-gamma levels produced by immune T cells in response to restimulation with target antigen in vitro. In addition, mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2. Thus, the combination vaccine regimen delivered by rVCG elicited adequate immune effectors that simultaneously protected against the individual pathogens.     

HIV Malaria Co-Infection Is Associated with Atypical Memory B Cell Expansion and a Reduced Antibody Response to a Broad Array of Plasmodium falciparum Antigens in Rwandan Adults

HIV infected individuals in malaria endemic areas experience more frequent and severe malaria episodes compared to non HIV infected. This clinical observation has been linked to a deficiency in antibody responses to Plasmodium falciparum antigens; however, prior studies have only focused on the antibody response to <0.5% of P. falciparum proteins. To obtain a broader and less-biased view of the effect of HIV on antibody responses to malaria we compared antibody profiles of HIV positive (HIV+) and negative (HIV-) Rwandan adults with symptomatic malaria using a microarray containing 824 P. falciparum proteins. We also investigated the cellular basis of the antibody response in the two groups by analyzing B and T cell subsets by flow cytometry. Although HIV malaria co-infected individuals generated antibodies to a large number of P. falciparum antigens, including potential vaccine candidates, the breadth and magnitude of their response was reduced compared to HIV- individuals. HIV malaria co-infection was also associated with a higher percentage of atypical memory B cells (MBC) (CD19+CD10-CD21-CD27-) compared to malaria infection alone. Among HIV+ individuals the CD4⁺ T cell count and HIV viral load only partially explained variability in the breadth of P. falciparum-specific antibody responses. Taken together, these data indicate that HIV malaria co-infection is associated with an expansion of atypical MBCs and a diminished antibody response to a diverse array of P. falciparum antigens, thus offering mechanistic insight into the higher risk of malaria in HIV+ individuals.     

Diversity of microbial sialic acid metabolism.

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.