Adenosine 5'-diphosphate-dependent subunit dissociation of bovine dopamine beta-hydroxylase

In a previous study, it was shown that purified soluble bovine dopamine beta-hydroxylase exhibits pH-dependent reversible tetramer-dimer dissociation (Saxena, A., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1985) J. Biol. Chem. 260, 3386-3392). Here we report evidence for the dissociation of this enzyme by magnesium-adenosine diphosphate independent of pH in the pH range 5-7. Quantitative binding of ADP to dopamine beta-hydroxylase revealed that there are two binding sites/dimeric species of hydroxylase and that ADP is tightly bound with a KD less than 10(-8) M. Kinetic data obtained at pH 5.5, the pH inside the chromaffin granule, shows that the apparent Km values for both the substrates tyramine and ascorbate are lowered by the presence of ADP without affecting the Vmax of the enzyme. The ADP-dependent lowering of apparent Km values results from a dissociation of the enzyme to the dimeric species which has inherently lower apparent Km values for substrates.     

DNA structural polymorphism modulates the kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

A compartmental model developed by Hensley (Hensley, P., Nardone, G., Chirikjian, J.G., and Wastney, M. E., (1990) J. Biol. Chem. 265, 15300-15307) for analysis of the time courses of the cleavage of superhelical DNA substrates by the restriction endonuclease, BamHI, has been used to quantify the effects of changes in temperature, ionic strength, superhelical density, and the DNA substrate on the binding and strand cleavage processes. Studies reported here indicate that changes in topology may be introduced into the DNA substrate solely as a result of the plasmid preparation process and in the absence of covalent bond cleavage and ligation. These changes in topology have qualitatively different effects on the kinetics than those promoted by changes in the superhelical density. The former are removed by briefly warming the DNA prior to assay, suggesting that they are only kinetically stable, while the latter changes are not affected by heating. Increasing the [NaCl] from 0.01 M to 0.1 M increases the overall rate of plasmid cleavage by increasing both the rates of cleavage and enzyme DNA association. To describe the decrease in the overall cleavage rate observed in 0.15 M NaCl, an ionic strength-dependent rate-determining structural transition in the DNA substrate was incorporated into the model. The largest changes in the rate of the cleavage process resulted from changes in the DNA substrate. For the SV40 substrate compared to pBR322, the rate constants describing the two association processes and the first bond cleavage event were increased 6- to 7-fold. The rate of the second bond cleavage process was not affected. These changes may be due to differences in the flanking sequences.     

Base pairing between hepatitis C virus RNA and microRNA 122 3' of its seed sequence is essential for genome stabilization and production of infectious virus

MicroRNA 122 (miR-122) facilitates hepatitis C virus (HCV) replication by recruiting an RNA-induced silencing complex (RISC)-like complex containing argonaute 2 (Ago2) to the 5' end of the HCV genome, thereby stabilizing the viral RNA. This requires base pairing between the miR-122 "seed sequence" (nucleotides [nt] 2 to 8) and two sequences near the 5' end of the HCV RNA: S1 (nt 22 to 28) and S2 (nt 38 to 43). However, recent reports suggest that additional base pair interactions occur between HCV RNA and miR-122. We searched 606 sequences from a public database (genotypes 1 to 6) and identified two conserved, putatively single-stranded RNA segments, upstream of S1 (nt 2 and 3) and S2 (nt 30 to 34), with potential for base pairing to miR-122 (nt 15 and 16 and nt 13 to 16, respectively). Mutagenesis and genetic complementation experiments confirmed that HCV nt 2 and 3 pair with nt 15 and 16 of miR-122 bound to S1, while HCV nt 30 to 33 pair with nt 13 to 16 of miR-122 at S2. In genotype 1 and 6 HCV, nt 4 also base pairs with nt 14 of miR-122. These 3' supplementary base pair interactions of miR-122 are functionally important and are required for Ago2 recruitment to HCV RNA by miR-122, miR-122-mediated stabilization of HCV RNA, and production of infectious virus. However, while complementary mutations at HCV nt 30 and 31 efficiently rescued the activity of a 15C,16C miR-122 mutant targeting S2, similar mutations at nt 2 and 3 failed to rescue Ago2 recruitment at S1. These data add to the current understanding of miR-122 interactions with HCV RNA but indicate that base pairing between miR-122 and the 5' 43 nt of the HCV genome is more complex than suggested by existing models.     

A survey of sesamin and composition of tocopherol variability from seeds of eleven diverse sesame (Sesamum indicum L.) genotypes using HPLC-PAD-ECD

The objective of this study was to determine the composition and content of sesamin and desmethyl tocopherols such as alpha-tocopherol (alphaT), delta-tocopherol (deltaT) and gamma-tocopherol (gammaT) in seeds of sesame (Sesamum indicum L.) for 11 genotypes conserved in the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia, USA. Seed accessions studied were collections from eight countries worldwide, including one landrace from Thailand and two cultivars from Texas, USA. Novel methodologies and analytical techniques described herein consisted of reverse-phase high-performance liquid chromatography (HPLC) connected in series with two detection systems specific for each analyte class. Photodiode array detection was employed for sesamin analysis and electrochemical array detection was used in the determination of tocopherols. A preliminary study was conducted to assess sesamin levels in 2003 and tocopherol levels in 2004 from sesame seed samples conserved at the USDA, ARS and PGRCU. In 2005, sesame seed samples were grown, harvested and evaluated for sesamin as well as tocopherol levels. The overall results (n = 3) showed that sesamin, alphaT, deltaT and gammaT levels were 0.67-6.35 mg/g, 0.034-0.175 microg/g, 0.44-3.05 microg/g and 56.9-99.3 microg/g respectively, indicating that the sesame seed accessions contained higher levels of sesamin and gammaT compared with alphaT and deltaT. Statistical analysis was conducted and significant differences were observed among the 11 different sesame genotypes. This suggests that genetic, environmental and geographical factors influence sesamin and desmethyl tocopherol content.     

The pigtail macaque (Macaca nemestrina) model of COVID-19 reproduces diverse clinical outcomes and reveals new and complex signatures of disease

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over five million people worldwide as of December 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics.     

Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.     

Reactive oxygen species in choline deficiency induced carcinogenesis and nitrone inhibition

Reactive oxygen species and free radical processes have been considered important in cancer development for many years. Much research demonstrates that the choline-deficiency induced hepatocarcinogenesis model prominently involves reactive oxygen species. We present a summary of results obtained in our original studies of this model over the last 4 years. We have shown that -phenyl-tert-butyl nitrone (PBN) and some of its hydroxylated derivatives (the 4- and 3-hydroxylated compounds) prevent hepatocarcinogenesis in this model. Mechanistic studies have demonstrated that isolated mitochondria from the livers of rats fed the choline-deficiency defined amino acid diet produce significantly much more H₂O₂ per NADH reducing equivalents oxidized. Based on these observations, we postulate that H₂O₂ is a primary carcinogenic factor in this model. Based on studies of the action of PBN on isolated mitochondria, we postulate that the inhibiting action of PBN involves suppression of H₂O₂ production of mitochondria and generally decreasing the oxidative stress within the preneoplastic lesions. The net effect of the activity of the nitrone compounds appears to be due to their ability to shift the apoptosis/neoplastic tendency balance toward apoptosis of the cells within the preneoplastic lesions. This is considered to be the primary reason the size of the preneoplastic lesions are significantly decreased and why the nitrones are potent anti-carcinogenic agents in this model.     

The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever

Background  

Infection with Ebola virus (EBOV) causes a fulminant and often fatal hemorrhagic fever. In order to improve our understanding of EBOV pathogenesis and EBOV-host interactions, we examined the molecular features of EBOV infection in vivo.     

Redox proteomics analysis of oxidatively modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease characterized by the loss of neuronal function in the motor cortex, brain stem, and spinal cord. Familial ALS cases, accounting for 10-15% of all ALS disease, are caused by a gain-of-function mutation in Cu,Zn-superoxide dismutase (SOD1). Two hypotheses have been proposed to explain the toxic gain of function of mutant SOD (mSOD). One is that mSOD can directly promote reactive oxygen species and reactive nitrogen species generation, whereas the other hypothesis suggests that mSODs are prone to aggregation due to instability or association with other proteins. However, the hypotheses of oxidative stress and protein aggregation are not mutually exclusive. G93A-SOD1 transgenic mice show significantly increased protein carbonyl levels in their spinal cord from 2 to 4 months and eventually develop ALS-like motor neuron disease and die within 5-6 months. Here, we used a parallel proteomics approach to investigate the effect of the G93A-SOD1 mutation on protein oxidation in the spinal cord of G93A-SOD1 transgenic mice. Four proteins in the spinal cord of G93A-SOD1 transgenic mice have higher specific carbonyl levels compared to those of non-transgenic mice. These proteins are SOD1, translationally controlled tumor protein (TCTP), ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1), and, possibly, alphaB-crystallin. Because oxidative modification can lead to structural alteration and activity decline, our current study suggests that oxidative modification of UCH-L1, TCTP, SOD1, and possibly alphaB-crystallin may play an important role in the neurodegeneration of ALS.