Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

1H nuclear magnetic resonance study of the prosthetic group in sulfhemoglobin.

The molecular and electronic structure of the modified prosthetic group of sulfhemoglobin (SHb) was investigated by 1H NMR for the low-spin ferric cyano-met and high-spin ferrous deoxy sulfhemoglobin complex. The 1H NMR resonances of the two subunits in the cyano-met SHb complex were differentiated on the basis of the differential stability toward regeneration of native subunits. The subunit origin for the two sets of resonances was established by formation of the sulfglobin protein for the isolated alpha-chain prior to assembling with the native beta-subunit to yield a tetramer with sulfhemin in the alpha-subunits. The subunit peak assignments establish that it is the beta-subunit of SHb which regenerates more rapidly to native protein. The hyperfine shifted sulfhemin peaks were assigned based on steady-state nuclear Overhauser effects which demonstrated that similarly hyperfine shifted peaks exhibit the same dipolar connectivities observed in the analogous sulfmyoglobin complex. Hence it is concluded that pyrrole B is the site of reaction in both hemoglobin and myoglobin. The initially formed SHb complex failed to equilibrate to yield a complex with a sulfhemin sufficiently stable to extraction as found previously for sulfmyoglobin. However, apoHb readily bound the green sulfhemin extracted from the terminal alkaline equilibration product of sulfmyoglobin. The inhibition on the equilibration to the alkaline form with the exocyclic thiolene ring is attributed to the interaction with Val FG5. The observations of the same dipolar connectivities among similarly hyperfine shifted peaks in the directly prepared and reconstituted SHb complexes further support the same structure for the sulfhemin in sulfmyoglobin and SHb. The strongly hyperfine shifted peaks in the deoxy form of both SHb complexes were found very similar to those of the analogous sulfmyoglobin complexes. The proximal His labile ring proton signal appears to experience a 5- to 10-ppm decrease upon conversion of a native globin to sulfglobin. This attenuation may provide a probe for differentiating chlorins and hemins in globin pockets.     

The development of oral vaccines based on live attenuated Salmonella strains

Abstract Safe, live attenuated Salmonella strains can be produced by introducing defined non-reverting mutations into the chromosome. Such rationally attenuated strains have proved to be excellent oral vaccines in several animal species and can therefore be considered as candidate vaccines against invasive salmonellosis in both animals and man. A panel of attenuating lesions is now available from which it is possible to tailor the level of attenuation and hence produce strains with different immunogenic properties. Because of the spectrum of immune responses produced by such Salmonella vaccine strains they have been utilised extensively as vectors for delivering heterologous antigens to the mammalian immune system. We have focussed on the development of a single dose oral tetanus vaccine based on attenuated Salmonella strains expressing a non-toxic, immunogenic protein derived from tetanus toxin (fragment C). Several different expression systems have been used for fragment C and candidate vaccine strains have been constructed that are capable of protecting orally immunised mice against a lethal challenge with tetanus toxin. An oral tetanus vaccine may help to reduce the mortality rate from tetanus in the developing world by overcoming the problems associated with the implementation of vaccine programmes using the current parenteral vaccine.     

The involvement of tumor necrosis factor in immunity to Salmonella infection

The role of TNF in immunity to Salmonella in mice was studied. Antiserum specific for murine TNF was raised and used to neutralize TNF activity in vivo. Injection of this serum into mice infected with the moderately mouse virulent Salmonella typhimurium strain M525 caused exacerbation of disease. Such treatment had no effect on the course of an infection with an attenuated S. typhimurium aroA (strain SL3261) mutant. However, the protection afforded by immunisation with live SL3261 against challenge with the virulent parent strain (SL1344) was abolished by anti-TNF antiserum. Interestingly both early (3 wk) immunity and late (10 wk) immunity was neutralized by such treatment. Inasmuch as early immunity is considered to be nonspecific and macrophage-mediated while late immunity is considered to be serotype-specific and T cell mediated, this suggests that TNF plays a role in protection from Salmonellosis in both cases.     

Expression of human IL-1 beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo.

The feasibility of using Salmonella typhimurium aroA mutant (SL3261) to deliver protein therapeutic agents was investigated in a murine model system. We have constructed an Escherichia coli expression plasmid designed to express the human protein IL-1 beta. This plasmid expresses IL-1 beta to high levels (greater than 30% total cell protein) in E. coli. In Salmonella the IL-1 beta is expressed constitutively to about 10% total cell protein, as verified by Western blotting analysis using polyclonal rabbit anti-IL-1 beta antibody. The protein is produced in a soluble and biologically active form. BALB/c mice administered orally or i.v. with S. typhimurium aroA mutants carrying the plasmid produced highly significant antibody responses against human IL-1 beta as determined by a solid-phase RIA. Furthermore, mice injected with the construct were significantly protected against lethal gamma-irradiation (850 rad). This study therefore demonstrates that the vaccine strain of Salmonella mutants can also be used effectively to deliver therapeutic proteins in vivo.     

Genomic regions associated with microdeletion/microduplication syndromes exhibit extreme diversity of structural variation

Segmental duplications (SDs) are a class of long, repetitive DNA elements whose paralogs share a high level of sequence similarity with each other. SDs mediate chromosomal rearrangements that lead to structural variation in the general population as well as genomic disorders associated with multiple congenital anomalies, including the 7q11.23 (Williams–Beuren Syndrome, WBS), 15q13.3, and 16p12.2 microdeletion syndromes. Population-level characterization of SDs has generally been lacking because most techniques used for analyzing these complex regions are both labor and cost intensive. In this study, we have used a high-throughput technique to genotype complex structural variation with a single molecule, long-range optical mapping approach. We characterized SDs and identified novel structural variants (SVs) at 7q11.23, 15q13.3, and 16p12.2 using optical mapping data from 154 phenotypically normal individuals from 26 populations comprising five super-populations. We detected several novel SVs for each locus, some of which had significantly different prevalence between populations. Additionally, we localized the microdeletion breakpoints to specific paralogous duplicons located within complex SDs in two patients with WBS, one patient with 15q13.3, and one patient with 16p12.2 microdeletion syndromes. The population-level data presented here highlights the extreme diversity of large and complex SVs within SD-containing regions. The approach we outline will greatly facilitate the investigation of the role of inter-SD structural variation as a driver of chromosomal rearrangements and genomic disorders.     

Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite,Caloric Intake and Fat Storage in Drosophila

Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C_14:0) and ceramide diene (C_14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C_14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.     

The magic bullet?

The promise of gene therapy has yet to be realised and a recent death has cast a long shadow over clinical trials. Will gene therapy develop into the magic bullet of the 21st Century or is it a technology too complicated to succeed?     

The hormonal regulation of axillary bud growth in Arabidopsis

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.     

Role of calpains in diabetes mellitus: A mini review

Type 2 diabetes mellitus (T2DM) is characterized by defects in haepatic glucose production, insulin action and insulin secretion, which can also lead to a variety of secondary disorders. The disease can lead to death without treatment and it has been predicted that T2DM will affect 215 million people world-wide by 2010. T2DM is a multifactorial condition whose precise genetic causes and biochemical defects have not been fully elucidated but at both levels, calpains appear to play a role. Positional cloning studies mapped T2DM susceptibility to CAPN10, the gene encoding the intracellular cysteine protease, calpain 10. Further studies have shown a number of non-coding polymorphisms in CAPN10 to be functionally associated with T2DM whilst the identification of coding polymorphisms, suggested that mutant calpain 10 proteins may also contribute to the disease. The presence of both calpain 10 and its mRNA have been demonstrated in tissues from several mammalian species whilst calpain 10 appears to be associated with pathways involved in glucose metabolism, insulin secretion and insulin action. It appears that other calpains may also participate in these pathways and here we present an overview of recent studies on calpains and their putative role in T2DM. (Mol Cell Biochem 261: 161–167, 2004)