Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania.

For many years, mRNA abundance has been used as the surrogate measure of gene expression in biological systems. However, recent genome-scale analyses in both bacteria and eukaryotes have revealed that mRNA levels correlate with steady-state protein abundance for only 50-70% of genes, indicating that translation and post-translation processes also play important roles in determining gene expression. What is not yet clear is whether dynamic processes such as cell cycle progression, differentiation, or response to environmental changes change the relationship between mRNA and protein abundance. Here, we describe a systems approach to interrogate promastigote-to-amastigote differentiation in the obligatory intracellular parasitic protozoan Leishmania donovani. Our results indicate that regulation of mRNA levels plays a major role early in the differentiation process, while translation and post-translational regulation are more important in the latter part. In addition, it appears that the differentiation signal causes a transient global increase in the rate of protein synthesis, which is subsequently down-regulated by phosphorylation of α-subunit of translation initiation factor 2. Thus, Leishmania dynamically changes the relationship between mRNA and protein abundance as it adapts to new environmental circumstances. It is likely that similar mechanisms play a more important role than previously recognized in regulation of gene expression in other organisms.

An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia, an economically devastating rickettsial disease of farmed salmonids. Infected salmonids respond poorly to antibiotic treatment and no effective vaccine is available for the control of P. salmonis. Bacterin preparations of P. salmonis were found to elicit a dose-dependent response in coho salmon (Oncorhynchus kisutch), which varied from inadequate protection to exacerbation of the disease. However, an outer surface lipoprotein of P. salmonis, OspA, recombinantly produced in Escherichia coli elicited a high level of protection in vaccinated coho salmon with a relative percent survival as high as 59% for this single antigen. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein, that are universally immunogenic in mammalian immune systems, were incorporated tandemly into an OspA fusion protein. Addition of these TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a three-fold increase in vaccine efficacy. These results represent a highly effective monovalent recombinant subunit vaccine for a rickettsia-like pathogen, P. salmonis, and for the first time demonstrate the immunostimulatory effect of mammalian TCE's in the salmonid immune model. These results may also be particularly pertinent to salmonid aquaculture in which the various subspecies are outbred and of heterologous haplotypes.

OspA, a lipoprotein antigen of the obligate intracellular bacterial pathogen Piscirickettsia salmonis.

No effective recombinant vaccines are currently available for any rickettsial diseases. In this regard the first non-ribosomal DNA sequences from the obligate intracellular pathogen Piscirickettsia salmonis are presented. Genomic DNA isolated from Percoll density gradient purified P. salmonis, was used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal antiserum raised against P. salmonis, with a bias toward P. salmonis surface antigens, was used to identify immunoreactive clones. Catabolite repression of the lac promoter was required to obtain a stable clone of a 4,983 bp insert in Escherichia coli due to insert toxicity exerted by the accompanying radA open reading frame (ORF). DNA sequence analysis of the insert revealed 1 partial and 4 intact predicted ORF's. A 486 bp ORF, ospA, encoded a 17 kDa antigenic outer surface protein (OspA) with 62% amino acid sequence homology to the genus common 17 kDa outer membrane lipoprotein of Rickettsia prowazekii, previously thought confined to members of the genus Rickettsia. Palmitate incorporation demonstrated that OspA is posttranslationally lipidated in E. coli, albeit poorly expressed as a lipoprotein even after replacement of the signal sequence with the signal sequence from lpp (Braun lipoprotein) or the rickettsial 17 kDa homologue. To enhance expression, ospA was optimized for codon usage in E. coli by PCR synthesis. Expression of ospA was ultimately improved (approximately 13% of total protein) with a truncated variant lacking a signal sequence. High level expression (approximately 42% tot. prot.) was attained as an N-terminal fusion protein with the fusion product recovered as inclusion bodies in E. coli BL21. Expression of OspA in P. salmonis was confirmed by immunoblot analysis using polyclonal antibodies generated against a synthetic peptide of OspA (110-129) and a strong antibody response against OspA was detected in convalescent sera from coho salmon (Oncorhynchus kisutch).

Fundamental limits on third-order molecular susceptibilities.

By the use of sum rules, the largest third-order molecular nonlinear-optical susceptibilities allowed by quantum mechanics are determined. The theoretical upper limit is found to depend only on the first excited-state transition energy and on the number of electrons, in agreement with experimental data.

Fabrication and characterization of single-mode electro-optic polymer optical fiber.

We report on what we believe is the first demonstration of single-mode polymer optical fiber with embedded electrodes. We show that the electrodes can be used to pole the dye-doped core and to electro-optically phase modulate the light in the waveguide.

Suppressing vibrations in a sheet with a Fabry-Perot photomechanical device.

We report what we believe to be the first demonstration that the vibration amplitude in a stretched plastic sheet can be reduced by as much as 10 dB with a monolithic all-optical mesoscale photomechanical device.

Antigenic characterization of the salmonid pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis, the etiological agent of salmonid rickettsial septicemia, was purified from infected immortal chinook salmon (Oncorhynchus tshawytscha) embryo cells by a combination of differential and Percoll density gradient centrifugation. Immune sera from rabbits immunized with purified whole cells of P. salmonis reacted with four protein antigens and two carbohydrate antigens with relative molecular sizes of 65, 60, 54, 51, 16, and approximately 11 kDa, respectively. The carbohydrate antigens appear to be mainly core region lipo-oligosaccharide with lesser amounts of lipopolysaccharide. Serum from convalescent rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) reacted with several minor immunoreactive protein antigens between 10 and 70 kDa in size and a carbohydrate antigen with a relative molecular size of approximately 11 kDa. The salmonid immune system did not appear to elicit a strong humoral response against this intracellular pathogen. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis cells suggest that the immunoreactive antigens identified with rabbit antisera are surface exposed and differ significantly from those identified with salmonid antisera.

Sagnac interferometric intensity-dependent refractive-index measurements of polymer optical fiber.

We have applied a new modified Sagnac interferometric technique to measure the real part of the intensitydependent refractive index of a single-mode polymer optical fiber. For a 0.1% by weight squaraine dye in a poly(methyl methacrylate) core, Re([chi((3))1111]) is 12(+/-7) x 10(-13) cm(3)/erg at lambda = 1064 nm. We discuss the effect of these measurements on all-optical devices.

Measurement of ultrafast optical nonlinearities using a modified Sagnac interferometer.

A method for the measurement of fast, intensity-dependent refractive-index changes with the use of a modified Sagnac ring interferometer is presented. The measurement is not degraded by slowly responding background index changes. Nonlinear refractive-index changes in an undoped silicon wafer, and in poly-bis toluene sulfonate polydiacetylene and dye-doped polymethyl methacrylate waveguides, were measured with the use of a cw mode-locked Nd:YAG laser.