Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Nerve Growth Factor Administration Attenuates Cognitive but Not Neurobehavioral Motor Dysfunction or Hippocampal Cell Loss Following Fluid-Percussion Brain Injury in Rats

Abstract: Lateral fluid-percussion brain injury in rats results in cognitive deficits, motor dysfunction, and selective hippocampal cell loss. Neurotrophic factors have been shown to have potential therapeutic applications in neurodegenerative diseases, and nerve growth factor (NGF) has been shown to be neuroprotective in models of excitotoxicity. This study evaluated the neuroprotective efficacy of intracerebral NGF infusion after traumatic brain injury. Male Sprague-Dawley rats received lateral fluid-percussion brain injury of moderate severity (2.1–2.3 atm). A miniosmotic pump was implanted 24 h after injury to infuse NGF (n = 34) or vehicle (n = 16) directly into the region of maximal cortical injury. Infusions of NGF continued until the animal was killed at 72 h, 1 week, or 2 weeks after injury. Animals were evaluated for cognitive dysfunction (Morris Water Maze) and regional neuronal cell loss (Nissl staining) at each of the three time points. Animals surviving for 1 or 2 weeks were also evaluated for neurobehavioral motor function. Although an improvement in memory scores was not observed at 72 h after injury, animals receiving NGF infusions showed significantly improved memory scores when tested at 1 or 2 weeks after injury compared with injured animals receiving vehicle infusions ( p < 0.05). Motor scores and CA3 hippocampal cell loss were not significantly different in any group of NGF-treated animals when compared with controls. These data suggest that NGF administration, in the acute, posttraumatic period following fluid-percussion brain injury, may have potential in improving post-traumatic cognitive deficits.     

Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease

-Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalyticmechanism, structure–function relationships, and more recently forstudies of pro region-assisted protein folding. We have assigned thebackbones of the enzyme alone, and of its complex with the tetrahedraltransition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, usingdouble- and triple-resonance 3D NMR spectroscopy on uniformly¹⁵N- and ¹³C/¹⁵N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibitedform of the protein are correlated with distance from the inhibitor, thedisplacement of backbone nitrogens, and change in hydrogen bond strengthupon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzymewith that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, wasobserved in many of the active site amides (including three broadened toinvisibility), and in a majority of cases the broadening was reversed uponaddition of the inhibitor. Implications and possible mechanisms of this linebroadening are discussed.     

Does PBAN play an alternative role of controlling pheromone emission in the cabbage looper moth,Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae)?

There is an active process by which sex pheromone reserves of female cabbage looper moths, Trichoplusia ni, are transported to the gland's surface during the nocturnal period of calling. We hypothesized that this mobilization was controlled by a head factor, possibly related to the pheromone biosynthesis activating neuropeptides (PBAN) that in other species stimulate pheromone synthesis. We evaluated the impact of head extracts of T. ni on pheromone emission and glandular content of pheromone. During the photophase injected head extracts stimulated an increased pheromone emission rate in females, but glandular content of pheromone was not affected. Head extracts of H. virescens, a species with known PBAN activity, and synthetic PBAN stimulated an increased pheromone emission rate in T. ni. There was some specificity of the response of female T. ni to PBAN, in that several other unrelated polypeptides did not stimulate this type of response. Previously it had been determined that brain factors do not play a role in stimulating pheromone biosynthesis in T. ni. Our results indicate that there may be additional avenues by which PBAN or related neuropeptides control pheromone emission, including transport of pheromone reserves to the surface of the sex pheromone gland.     

Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin.

Inoculation of mice with hemagglutinin (HA)-expressing DNA affords reliable protection against lethal influenza virus infection, while in chickens the same strategy has yielded variable results. Here we show that gene gun delivery of DNA encoding an H5 HA protein confers complete immune protection to chickens challenged with lethal H5 viruses. In tests of the influence of promoter selection on vaccine efficacy, close correlations were obtained between immune responses and the dose of DNA administered, whether a cytomegalovirus (CMV) immediate-early promoter or a chicken beta-actin promoter was used. Perhaps most important, the HA-DNA vaccine conferred 95% cross-protection against challenge with lethal antigenic variants that differed from the primary antigen by 11 to 13% (HA1 amino acid sequence homology). Overall, the high levels of protection seen with gene gun delivery of HA-DNA were as good as, if not better than, those achieved with a conventional whole-virus vaccine, with fewer instances of morbidity and death. The absence of detectable antibody titers after primary immunization, together with the rapid appearance of high titers immediately after challenge, implicates efficient B-cell priming as the principal mechanism of DNA-mediated immune protection. Our results suggest that the efficacy of HA-DNA influenza virus vaccine in mice extends to chickens and probably to other avian species as well. Indeed, the H5 preparation we describe offers an attractive means to protect the domestic poultry industry in the United States from lethal H5N2 viruses, which continue to circulate in Mexico.     

Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta.