Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Rapid screening for metabolite overproduction in fermentor broths, using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks

Binary mixtures of model systems consisting of the antibiotic ampicillin with either Escherichia coli or Staphylococcus auresu were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra, so as to obtain quantitative information on the concentration of ampicilin in the mixtures, partial least squares regression (PLS), principal components regression (PCR), and fully interconnected feedforward artificial neural networks (ANNs) were studied. In the latter case, the weights were modified using the standard backpropagation algorithm, and the nodes used a sigmoidal squsahing funciton. It was found that each of the methods could be used to provide calibration models which gave excellent predictions for the concentrations of ampicillin in samples on which they had not been trained. Furthermore, ANNs trained to predict the amount of ampicilin in E. coli were able to generalise so as to predict the concentration of ampicillin in a S. aureus background, illustrating the robustness of ANNs to rather substantial variations in the biological background. The PyMS of the complex mixture of ampicilin in bacteria could not be expressed simply in terms of additive combinations of the spectra describing the pure components of the mixtures and their relative concentrations. Intermolecular reactions took place in the pyrolysate, leading to a lack of superposition of the spectral components and to a dependence of the normalized mass spectrum on sample size. Samples from fermentations of a single organism in a complex production medium were also analyzed quantitatively for a drug of commercial interest. The drug could also be quantified in a variety of mutant-producing strains cultivated in the same medium. The combination of PyMS and ANNs constitutes a novel, rapid, and convenient method for exploitation in strain improvement screening programs. (c) 1994 John Wiley & Sons, Inc.     

Pathogenesis of Aleutian mink disease parvovirus infection: effects of suppression of antibody response on viral mRNA levels and on development of acute disease.

We suppressed the B-cell development and antibody response in mink by using treatment with polyclonal anti-immunoglobulin M (anti-IgM) to study the effects of antiviral antibodies on development of Aleutian mink disease parvovirus (ADV)-induced disease in more detail. Newborn mink kits were injected intraperitoneally with 1 mg of either anti-IgM or a control preparation three times a week for 30 to 34 days. At 21 days after birth, groups of mink kits were infected with the highly virulent United isolate of ADV. At selected time points, i.e., postinfection days 9, 13, 29, and 200, randomly chosen mink kits were sacrificed, and blood and tissues were collected for analyses. The efficacy of immunosuppressive treatment was monitored by electrophoretic techniques and flow cytometry. Effects of treatment on viral replication, on viral mRNA levels, and on development of acute or chronic disease were determined by histopathological, immunoelectrophoretic, and molecular hybridization techniques. Several interesting findings emerged from these studies. First, antiviral antibodies decreased ADV mRNA levels more than DNA replication. Second, suppression of B-cell development and antibody response in mink kits infected at 21 days of age resulted in production of viral inclusion bodies in alveolar type II cells. Some of these kits showed mild clinical signs of respiratory disease, and one kit died of respiratory distress; however, clinical signs were seen only after release of immunosuppression, suggesting that the production of antiviral antibodies, in combination with the massive amounts of free viral antigen present, somehow is involved in the induction of respiratory distress. It is suggested that the antiviral antibody response observed in mink older than approximately 14 days primarily, by a yet unknown mechanism, decreases ADV mRNA levels which, if severe enough, results in restricted levels of DNA replication and virion production. Furthermore, such a restricted ADV infection at low levels paves the way for a persistent infection leading to immunologically mediated disease. The potential mechanisms of antibody-mediated restriction of viral mRNA levels and mechanisms of disease induction are discussed.     

Pathogenesis of adenovirus type 5 pneumonia in cotton rats (Sigmodon hispidus).

Cotton rats (Sigmodon hispidus) were inoculated intranasally with 10(2.0) to 10(10.0) PFU of human adenovirus type 5. The virus replicated to a high titer in pulmonary tissues, with the peak titer being proportional to the input dose. The 50% lethal dose was 10(9.4) PFU. Histopathologic changes were proportional to the infecting inoculum and included the infiltration of interstitial and intra-alveolar areas, moderate damage to bronchiolar epithelium, and cellular infiltration of peribronchiolar and perivascular regions. These changes could be divided into two phases: an early phase (affecting alveoli, bronchiolar epithelium, and peribronchiolar regions) with an infiltrate consisting primarily of monocytes-macrophages and neutrophils, with occasional lymphocytes, and a later phase (affecting peribronchiolar and perivascular regions) with an infiltrate consisting almost exclusively of lymphocytes. In both phases, the predominant process was the response of the host to infection, rather than direct viral damage to infected cells. An infecting inoculum of 10(8.0) PFU or larger caused severe damage to type II alveolar cells, which were swollen, showed a loss of lamellar bodies, and were surrounded by polymorphonuclear leukocytes and macrophages. No evidence of complete viral replication was found in type II alveolar cells.     

Bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory infections in infancy. No vaccine against this virus has yet been protective, and antiviral drugs have been of limited utility. Using the cotton rat model of HRSV infection, we examined bovine respiratory syncytial virus (BRSV), a cause of acute respiratory disease in young cattle, as a possible vaccine candidate to protect children against HRSV infection. Cotton rats were primed intranasally with graded doses of BRSV/375 or HRSV/Long or were left unprimed. Three weeks later, they were challenged intranasally with either BRSV/375, HRSV/Long (subgroup A), or HRSV/18537 (subgroup B). At intervals postchallenge, animals were sacrificed for virus titration and histologic evaluation. Serum neutralizing antibody titers were determined at the time of viral challenge. BRSV/375 replicated to low titers in nasal tissues and lungs. Priming with 10(5) PFU of BRSV/375 effected a 500- to 1,000-fold reduction in peak nasal HRSV titer and a greater than 1,000-fold reduction in peak pulmonary HRSV titer upon challenge with HRSV/Long or HRSV/18537. In contrast to priming with HRSV, priming with BRSV did not induce substantial levels of neutralizing antibody against HRSV and was associated with a delayed onset of clearance of HRSV upon challenge. Priming with BRSV/375 caused mild nasal and pulmonary pathology and did not cause exacerbation of disease upon challenge with HRSV/Long. Our findings suggest that BRSV may be a potential vaccine against HRSV and a useful tool for studying the mechanisms of immunity to HRSV.     

New high-toxicity mosquitocidal strains of Bacillus sphaericus lacking a 100-kilodalton-toxin gene.

Five new high-toxicity mosquitocidal strains of Bacillus sphaericus were isolated in Singapore. They all belong to phage group 8 and have binary toxin (51.4- plus 41.9-kDa) genes located on the chromosome but lack a 100-kDa-toxin gene. These strains of B. sphaericus constitute a new subgroup, as only two weakly toxic strains in phage group 8 have previously been described and all the known high-toxicity strains have both binary toxin and 100-kDa-toxin genes.     

Recovery of the coral Montastrea annularis in the Florida Keys after the 1987 Caribbean “bleaching event”

Many reef-building corals and other cnidarians lost photosynthetic pigments and symbiotic algae (zooxanthellae) during the coral bleaching event in the Caribbean in 1987. The Florida Reef Tract included some of the first documented cases, with widespread bleaching of the massive coral Montastrea annularis beginning in late August. Phototransects at Carysfort Reef showed discoloration of >90% of colonies of this species in March 1988 compared to 0% in July 1986; however no mortality was observed between 1986 and 1988. Samples of corals collected in February and June 1988 had zooxanthellae densities ranging from 0.1 in the most lightly colored corals, to 1.6x10⁶ cells/cm² in the darker corals. Minimum densities increased to 0.5x10⁶ cells/cm² by August 1989. Chlorophyll-a content of zooxanthellae and zooxanthellar mitotic indices were significantly higher in corals with lower densities of zooxanthellae, suggesting that zooxanthellar at low densities may be more nutrientsufficient than those in unbleached corals. Ash-free dry weight of coral tissue was positively correlated with zooxanthellae density at all sample times and was significantly lower in June 1988 compared to August 1989. Proteins and lipids per cm² were significantly higher in August 1989 than in February or June, 1988. Although recovery of zooxanthellae density and coral pigmentation to normal levels may occur in less than one year, regrowth of tissue biomass and energy stores lost during the period of low symbiont densities may take significantly longer.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.