The Biotransformation of Propylene to Propylene Oxide by Methylococcus capsulatus (Bath): 3. Reactivation of Inactivated Whole Cells to Give a High Productivity System

Methylococcus capsulatus (Bath) possesses methane monooxygenases (soluble - (sMMO) and particulate - (pMMO)) which are able to catalyse the epoxidation of propylene to propylene oxide. In a previous paper we have shown that the production of the epoxide caused a rapid inactivation of the bioconversion process (Stanley et al, 1992). This paper shows that cultures containing pMMO, inactivated by propylene oxide production, could be completely reactivated in the presence of growth substrates within 5 h after the removal of propylene oxide so long as the propylene oxide production rate was below 150 nmol min^-1 [mg dry weight cells]^-1. Reactivation under these conditions was detectable within 30 min of propylene oxide removal. On the other hand, cells inactivated by propylene oxide production rates in excess of 150 nmol min^-1 [mg dry weight]^-1 did not begin to recover activity within the 30 min period. Furthermore a lag period was observed before reactivation began which was dependent upon the initial production rate. Cultures possessing sMMO took twice as long to recover their activity compared with cells containing pMMO.

Reactivation of propylene oxide production could occur without growth, but the process required the presence of a carbon and energy source (methane or methanol), sulphur, nitrogen and oxygen, although copper (which is normally involved in pMMO activity) was not required. It was shown that de novo protein synthesis was required for reactivation of activity.

Production rates of 12 g 1^-1 d^-1 could be maintained for longer than three weeks in a single phase production process and rates up to 30 g 1^-1 d^-1 were achieved in a two stage process. Using Methylocystis parvus (OBBP) rates of up to 90 g 1^-1 d^-1 were attained over a one week period.     

Effectiveness of ascorbate and ascorbate peroxidase in promoting nitrogen fixation in model systems

Ascorbate and ascorbate peroxidase are important antioxidants that are abundant in N2-fixing legume root nodules. Antioxidants are especially critical in root nodules because leghemoglobin, which is present at high concentrations in nodules, is prone to autoxidation and production of activated oxygen species such as O2.- and H2O2. The merits of ascorbate and ascorbate peroxidase for maintaining conditions favorable for N2 fixation were examined in two model systems containing oxygen-binding proteins (purified myoglobin or leghemoglobin) and N2-fixing microorganisms (free-living Azorhizobium or bacteroids of Bradyrhizobium japonicum) in sealed vials. The inclusion of ascorbate alone to these systems led to enhanced oxygenation of hemeproteins, as well as to increases in nitrogenase (acetylene reduction) activity. The inclusion of both ascorbate and ascorbate peroxidase resulted in even greater positive responses, including increases of up to 4.5-fold in nitrogenase activity. In contrast, superoxide dismutase did not provide beneficial antioxidant action and catalase alone provided only very marginal benefit. Optimal concentrations were 2 mM for ascorbate and 200 micrograms/ml for ascorbate peroxidase. These concentrations are similar to those found in intact soybean nodules. These results support the conclusion that ascorbate and ascorbate peroxidase are beneficial for maintaining conditions favorable for N2 fixation in nodules.     

Mast cells impair the development of protective anti-tumor immunity

Mast cells have emerged as critical intermediaries in the regulation of peripheral tolerance. Their presence in many precancerous lesions and tumors is associated with a poor prognosis, suggesting mast cells may promote an immunosuppressive tumor microenvironment and impede the development of protective anti-tumor immunity. The studies presented herein investigate how mast cells influence tumor-specific T cell responses. Male MB49 tumor cells, expressing HY antigens, induce anti-tumor IFN-??⁺ T cell responses in female mice. However, normal female mice cannot control progressive MB49 tumor growth. In contrast, mast cell-deficient c-Kit^Wsh (W^sh) female mice controlled tumor growth and exhibited enhanced survival. The role of mast cells in curtailing the development of protective immunity was shown by increased mortality in mast cell-reconstituted W^sh mice with tumors. Confirmation of enhanced immunity in female W^sh mice was provided by (1) higher frequency of tumor-specific IFN-??⁺ CD8⁺ T cells in tumor-draining lymph nodes compared with WT females and (2) significantly increased ratios of intratumoral CD4⁺ and CD8⁺ T effector cells relative to tumor cells in W^sh mice compared to WT. These studies are the first to reveal that mast cells impair both regional adaptive immune responses and responses within the tumor microenvironment to diminish protective anti-tumor immunity.     

Transcript profiling and lipidomic analysis of ceramide subspecies in mouse embryonic stem cells and embryoid bodies

Ceramides (Cers) are important in embryogenesis, but no comprehensive analysis of gene expression for Cer metabolism nor the Cer amounts and subspecies has been conducted with an often used model: mouse embryonic stem cells (mESCs) versus embroid bodies (EBs). Measuring the mRNA levels by quantitative RT-PCR and the amounts of the respective metabolites by LC-ESI/MS/MS, notable differences between R1 mESCs and EBs were: EBs have higher mRNAs for CerS1 and CerS3, which synthesize C18- and C≥24-carbons dihydroceramides (DH)Cer, respectively; EBs have higher CerS2 (for C24:0- and C24:1-); and EBs have lower CerS5 + CerS6 (for C16-). In agreement with these findings, EBs have (DH)Cer with higher proportions of C18-, C24- and C26- and less C16-fatty acids, and longer (DH)Cer are also seen in monohexosylCers and sphingomyelins. EBs had higher mRNAs for fatty acyl-CoA elongases that produce C18-, C24-, and C26-fatty acyl-CoAs (Elovl3 and Elovl6), and higher amounts of these cosubstrates for CerS. Thus, these studies have found generally good agreement between genomic and metabolomic data in defining that conversion of mESCs to EBs is accompanied by a large number of changes in gene expression and subspecies distributions for both sphingolipids and fatty acyl-CoAs.     

Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Copper(I), copper(II) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus capsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron-sulphur centre of protein C with a secondary effect at the FAD centre when the copper(II):protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron-sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule. These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.     

Effect of Ischemia on the In Vivo Release of Striatal Dopamine, Glutamate, and γ-Aminobutyric Acid Studied by Intracerebral Microdialysis

We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesioning. The present study was carried out to evaluate whether the protective effect of the lesion involves changes in the degree of local cerebral blood flow (ICBF) reduction, energy metabolite depletion, or alterations in the extracellular release of striatal dopamine (DA), glutamate (Glu), or gamma-aminobutyric acid (GABA). Control and SN-lesioned rats were subjected to 20 min of forebrain ischemia by four-vessel occlusion combined with systemic hypotension. Levels of ICBF, as measured by the autoradiographic method, and energy metabolites were uniformly reduced in both the ipsi- and contralateral striata at the end of the ischemic period, a finding implying that the lesion did not affect the severity of the ischemic insult itself. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. An approximately 500-fold increase in DA content, a 7-fold increase in Glu content, and a 5-fold increase in GABA content were observed during ischemia in nonlesioned animals. These levels gradually returned to baseline by 30 min of reperfusion. In SN-lesioned rats, the release of DA was completely prevented, the release of GABA was not affected, and the release of Glu was partially attenuated. However, excessive extracellular Glu concentrations were still attained, which are potentially toxic. This, taken together with the previous neuropathological findings, suggests that excessive release of DA is important for the development of ischemic cell damage in the striatum.     

The Production of Catechols from Benzene and Toluene by Pseudomonas Putida in Glucose Fed-Batch Culture

Catechol and 3-methylcatechol were produced from benzene and toluene respectively using different mutants of Pseudomonas putida. P. putida 2313 lacked the extradiol cleavage enzyme, catechol 2,3-oxygenase, allowing overproduction of 3-methylcatechol from toluene to a level of 11.5 mM (1.27 g·1^-1) in glucose fed-batch culture. P. putida 6(12), a mutant of P. putida 2313, lacked both catechol-oxygenase and catechol 1,2-oxygenase, and accumulated catechol from benzene to a level of 27.5mM(3g·1^-1).

In both biotransformations product formation ceased within 10 hours of feeding the aromatic substrate, and this was due to product inhibition by the catechols. The primary site of catechol toxicity was inhibition of the aromatic dioxygenase. Neither cis-toluene dihydrodiol cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene), nor cis-benzene dihydrodiol (cis-l,2-dihydroxy-3-methylcyclohexa-3,5-diene) dehydrogenase was significantly inhibited by catechol overproduction whereas both ring activating dioxygenases were inhibited within 4-6 hours of the maximum product concentration being attained.

3-Methylcatechol overproduction from toluene was also studied using a continuous product removal system. Granular activated charcoal removed 3-methylcatechol efficiently and was easily regenerated by washing with ethyl acetate. Using P. putida 2313, it was shown that the final product concentration increased approximately fourfold. Additional products were formed and the significance of these are discussed.     

Acetylene as a suicide substrate and active site probe for methane monooxygenase from Methylococcus capsulatus (Bath)

Abstract Acetylene was shown to be an inhibitor of cell-free methane monooxygenase (MMO) activity in Methylococcus capsulatus (Bath). Inhibition was demonstrated for both the soluble and particulate forms of the enzyme and was dependent on the presence of both NADH and oxygen. Inactivation of the enzyme complex was irreversible and was due to binding of the acetylene to specific proteins of the enzyme complex. The use of radiolabelled [¹⁴C]acetylene provided a method for visualisation of the bound inhibitor: protein complex on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Acetylene was shown to bind to proteins which are associated with methane-oxidising activity and it is proposed that acetylene acts as a suicide substrate.     

Genetic assessment of an isolated endemic Samango monkey (<Emphasis Type="Italic">Cercopithecus albogularis labiatus</Emphasis>) population in the Amathole Mountains,Eastern Cape Province,South Africa

The endemic Samango monkey subspecies (Cercopithecus albogularis labiatus) inhabits small discontinuous Afromontane forest patches in the Eastern Cape, KwaZulu-Natal midlands and southern Mpumalanga Provinces in South Africa. The subspecies is affected by restricted migration between forest patches which may impact on gene flow resulting in inbreeding and possible localized extinction. Current consensus, based on habitat quality, is that C. a. labiatus can be considered as endangered as the small forest patches they inhabit may not be large enough to sustain them. The aim of this study was to conduct a molecular genetic investigation to determine if the observed isolation has affected the genetic variability of the subspecies. A total of 65 Samango monkeys (including juveniles, subadults and adults) were sampled from two localities within the Hogsback area in the Amathole Mountains. Nuclear and mitochondrial DNA variation was assessed using 17 microsatellite markers and by sequencing the hypervariable 1 region (HVR1). Microsatellite data generated was used to determine population structure, genetic diversity and the extent of inbreeding. Sequences of the HVR1 were used to infer individual origins, haplotype sharing and haplotype diversity. No negative genetic factors associated with isolation such as inbreeding were detected in the two groups and gene flow between groups can be regarded as fairly high primarily as a result of male migration. This was in contrast to the low nuclear genetic diversity observed (H _o = 0.45). A further reduction in heterozygosity may lead to inbreeding and reduced offspring fitness. Translocations and establishment of habitat corridors between forest patches are some of the recommendations that have emerged from this study which will increase long-term population viability of the subspecies.