Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion

We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile. These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes.     

Mutational analysis of a sequence-specific ssDNA binding lupus autoantibody

11F8 is a murine anti-ssDNA monoclonal autoantibody isolated from a lupus prone autoimmune mouse. This mAb binds sequence specifically, and prior studies have defined the thermodynamic and kinetic basis for sequence-specific recognition of ssDNA (Ackroyd, P. C., et al. (2001) Biochemistry 40, 2911-2922; Beckingham, J. A. and Glick, G. D. (2001) Bioorg. Med. Chem. 9, 2243-2252). Here we present experiments designed to identify the residues on 11F8 that mediate sequence-specific, noncognate, and nonspecific recognition of ssDNA and their contribution to the overall binding thermodynamics. Site-directed mutagenesis of an 11F8 single-chain construct reveals that six residues within the complementarity determining regions of 11F8 account for ca. 80% of the binding free energy and that there is little cooperativity between these residues. Germline-encoded aromatic and hydrophobic side chains provides the basis for nonspecific recognition of single-stranded thymine nucleobases. Sequence-specific recognition is controlled by a tyrosine in the heavy chain along with a somatically mutated arginine residue. Our data show that the manner in which 11F8 achieves sequence-specific recognition more closely resembles RNA-binding proteins such as U1A than other types of nucleic acid binding proteins. In addition, comparing the primary sequence of 11F8 with clonally related antibodies that differ by less than five amino acids suggests that somatic mutations which confer sequence specificity may be a feature that distinguishes glomerulotrophic pathogenic anti-DNA from those that are benign.     

A virulence attenuated amoebapore-less mutant of Entamoeba histolytica and its interaction with host cells

Entamoeba histolytica, the protozoan parasite which causes amoebiasis, is an exclusively human pathogen so developing a vaccine could effectively impact the spread of the disease. Recently we developed a genetically modified avirulent strain, termed G3, from the virulent E. histolytica strain HM-1:IMSS. The new strain lacks the important virulence factor, the amoebapore-A. The objective of our current study was to investigate the avirulence of the attenuated strain as well as to examine the antigenic and immunogenic responses of these trophozoites as potential candidates for a live vaccine. Functional assays were conducted to characterise the virulent behaviour of the G3 strain. This behaviour was compared to the virulent strain HM-1:IMSS and the non-virulent strain Rahman. Western blots were conducted to confirm the lack of amoebapore-A in the E. histolytica G3 strain and to demonstrate that it had no influence on the presence of other virulence factors. Results of these two sets of tests proved the G3 strain to be phenotypically similar to the avirulent Rahman strain while antigenically identical to the virulent HM-1:IMSS, apart from the lack of the amoebapore-A protein. Intraperitoneal immunisation of hamsters with G3 trophozoites compared to sham immunised hamsters resulted in IgG anti-HM-1:IMSS antibodies. The level of humoral response was variable and further testing has to take place before introducing this new strain as a vaccine.     

Vector for pop-in/pop-out gene replacement in Pichia pastoris

Gene replacement in yeast is often accomplished by using a counterselectable marker such as URA3. Although ura3 strains of Pichia pastoris have been generated, these strains are inconvenient to work with because they grow slowly, even in the presence of uracil. To overcome this limitation, we have developed an alternative counterselectable marker that can be used in any P. pastoris strain. This marker is the T-urf13 gene from the mitochondrial genome of male-sterile maize. Previous work showed that expression of a mitochondrially targeted form of T-urf13 in Saccharomyces cerevisiae rendered the cells sensitive to the insecticide methomyl, and similar results have now been obtained with P. pastoris. We have incorporated T-urf13 into a vector that also contains an ARG4 marker for positive selection. The resulting plasmid allows for pop-in/pop-out gene replacement in P. pastoris.     

Role of Pseudomonas putida indoleacetic acid in development of the host plant root system

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.     

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.     

Strategies used by rhizobia to lower plant ethylene levels and increase nodulation

Agriculture depends heavily on biologically fixed nitrogen from the symbiotic association between rhizobia and plants. Molecular nitrogen is fixed by differentiated forms of rhizobia in nodules located on plant roots. The phytohormone, ethylene, acts as a negative factor in the nodulation process. Recent discoveries suggest several strategies used by rhizobia to reduce the amount of ethylene synthesized by their legume symbionts, decreasing the negative effect of ethylene on nodulation. At least one strain of rhizobia produces rhizobitoxine, an inhibitor of ethylene synthesis. Active 1-aminocyclopropane-1-carboxylate (ACC) deaminase has been detected in a number of other rhizobial strains. This enzyme catalyzes the cleavage of ACC to alpha-ketobutyrate and ammonia. It has been shown that the inhibitory effect of ethylene on plant root elongation can be reduced by the activity of ACC deaminase.     

Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase?

The phenomenon of cold scission or cold lability, which entails a widespread variety of oligomeric enzymes, is still enigmatic. The effect of cooling on the activity and the quaternary structure of the pyridoxal 5'-phosphate (PLP)-dependent enzyme, tryptophanase (Tnase), was studied utilizing single photon counting time-resolved spectrofluorometry. Upon cooling of holo-wild-type (wt) Tnase and its W330F mutant from 25 degrees C to 2 degrees C, a reduction in PLP fluorescence lifetime and rotational correlation time as well as inactivation and dissociation from tetramers to dimers were observed for both enzymes. Fluorescence anisotropy invariably decreased as a consequence of cooling, whether it was accompanied by a slight decrease in activity without significant dissociation, or by a substantial decrease in activity that was associated with either a partial or major dissociation. These results support the suggested conformational change that precedes the PLP-aldimine bond scission. It is proposed that cold inactivation is initiated by the weakening of hydrophobic interactions, leading to conformational changes which are the driving force for the aldimine bond cleavage.