Flash photolysis-electron spin resonance studies of Photosystem I. A fast reduction component of P-700+

A 300 μs decay component of ESR Signal I (P-700⁺) in chloroplasts is observed following a 10 μs actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl₂). The fast transient reduction of P-700⁺ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl₂Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl₂. Oxidation-reduction measurements reveal that the fast P-700⁺ reduction component reflects electron transfer from a component with E_m = 375±10 mV (pH = 7.5). These data suggest the assignment of the 300-μs decay kinetics to electron transfer from cytochrome f (Fe²⁺) to P-700⁺, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171–1174 (1971)).     

Electron spin resonance investigations of mitochondrial electron transport in Neurospora crassa. Characterization of paramagnetic intermediates in a standard strain.

1. Submitochondrial particles from Neurospora strain inl-89601 have been analyzed by electron spin resonance spectroscopy (ESR). Numerous signals due to iron-sulfur proteins are observed at low temperatures. Analysis of these ESR signals at various temperatures allows the assignment of resonances to iron-sulfur centers 1-5 that have been described in other organisms. There are no discrepancies between the signals seen in Neurospora and those described in other organisms and it is likely that Neurospora mitochondria contain the same iron-sulfur centers that are observed elsewhere. 2. NADPH and NADH act to reduce the iron-sulfur centers of respiratory complex I. 3. The drug pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chlorphenyl)pyrrole] is an effective inhibitor of both NADH-supported and succinate-supported electron transport in Neurospora. 4. Analysis of pyrrolnitrin inhibition curves, respiration studies, ESR spectra, and the steady-state level of reduction of cytochrome b in the presence and absence of the drug shows that pyrrolnitrin acts to inhibit electron transport in Neurospora mitochondria at multiple sites in the region between ubiquinone and cytochrome b.     

A murine model of smoke inhalation

The United States has one of the world's largest per capita fire death rates. House fires alone kill >9,000 Americans annually, and smoke inhalation is the leading cause of mortality from structural fires. Animal models are needed to develop therapies to combat this problem. We have developed a murine model of smoke inhalation through the design, construction, and use of a controlled-environment smoke chamber. There is a direct relationship between the quantity of wood combusted and mortality in mice. As with human victims, the primary cause of death from smoke inhalation is an elevated blood carboxyhemoglobin level. Lethal (78%) and sublethal (50%) carboxyhemoglobin levels were obtained in mice subjected to varying amounts of smoke. Mice exposed to wood smoke demonstrated more dramatic pathology than mice exposed to cotton or polyurethane smoke. A CD-1 model of wood smoke exposure was developed, demonstrating type II cell hypertrophy, cytoplasmic blebbing, cytoplasmic vacuolization, sloughing, hemorrhage, edema, macrophage infiltration, and lymphocyte infiltration. The bronchoalveolar lavage fluid of smoke-exposed mice demonstrated a significant increase in total cell counts compared with those in control mice. These findings are comparable to the lung tissue response observed in human victims of smoke inhalation.     

A major site of bicarbonate effect in system II reaction. Evidence from ESR signal IIvf, fast fluorescence yield changes and delayed light emission.

In order to determine the major site of bicarbonate action in the electron transport complex of Photosystem II, the following experimental techniques were used: electron spin resonance measurements of Signal IIvf, measurements of chlorophyll a fluorescence yield rise and decay kinetics, and delayed light emission decay. From data obtained using these experimental techniques the following conclusions were made: (1) absence of bicarbonate causes a reversible inactivation of up to 40% of Photosystem II reaction center activity; (2) there is no significant effect of bicarbonate on electron flow from the charge accumulating S state to Z; (3) there is no significant effect of bicarbonate on electron flow from Z to P-680+; (4) electron flow from Q-- to the intersystem electron transport pool is inhibited by from 4- to 6-fold under bicarbonate depletion conditions.     

Altered Expression of Polycomb Group Genes in Glioblastoma Multiforme

The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.