Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.     

Responses of mammalian metaphase chromosomes to endonuclease digestion

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In ³H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.     

Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates.

Previous studies by Guntaka et al. have shown that the unintegrated DNA intermediates of avian RNA tumor virus replication can be readily isolated from cultures of the quail tumor line QT-6 at 1 day after infection. The intermediates include double-stranded linear and covalently closed circular DNA species. Using the analysis procedure of Southern together with previously obtained information regarding the sites of action of certain restriction endonucleases on avian sarcoma virus DNA, we have further characterized the viral DNA intermediates. Evidence is presented that, relative to the RNA genome, most of the linear species possess a direct terminal sequence redundancy equivalent to 0.5 X 10(6) +/- 0.3 X 10(6) daltons of double-stranded DNA. Some of the circular forms also possess a sequence redundancy of 0.21 X 10(6) +/- 0.03 X 10(6) daltons.     

Assessing “Dangerous Climate Change”: Required Reduction of Carbon Emissions to Protect Young People,Future Generations and Nature

We assess climate impacts of global warming using ongoing observations and paleoclimate data. We use Earth’s measured energy imbalance, paleoclimate data, and simple representations of the global carbon cycle and temperature to define emission reductions needed to stabilize climate and avoid potentially disastrous impacts on today’s young people, future generations, and nature. A cumulative industrial-era limit of ∼500 GtC fossil fuel emissions and 100 GtC storage in the biosphere and soil would keep climate close to the Holocene range to which humanity and other species are adapted. Cumulative emissions of ∼1000 GtC, sometimes associated with 2°C global warming, would spur “slow” feedbacks and eventual warming of 3–4°C with disastrous consequences. Rapid emissions reduction is required to restore Earth’s energy balance and avoid ocean heat uptake that would practically guarantee irreversible effects. Continuation of high fossil fuel emissions, given current knowledge of the consequences, would be an act of extraordinary witting intergenerational injustice. Responsible policymaking requires a rising price on carbon emissions that would preclude emissions from most remaining coal and unconventional fossil fuels and phase down emissions from conventional fossil fuels.     

Capsular Types of Klebsiella pneumoniae Revisited by wzc Sequencing

Capsule is an important virulence factor in bacteria. A total of 78 capsular types have been identified in Klebsiella pneumoniae. However, there are limitations in current typing methods. We report here the development of a new genotyping method based on amplification of the variable regions of the wzc gene. Fragments corresponding to the variable region of wzc were amplified and sequenced from 76 documented capsular types of reference or clinical strains. The remaining two capsular types (reference strains K15 and K50) lacked amplifiable wzc genes and were proven to be acapsular. Strains with the same capsular type exhibited ≧94% DNA sequence identity across the variable region (CD1-VR2-CD2) of wzc. Strains with distinct K types exhibited <80% DNA sequence identity across this region, with the exception of three pairs of strains: K22/K37, K9/K45, and K52/K79. Strains K22 and K37 shared identical capsular polysaccharide synthesis (cps) genes except for one gene with a difference at a single base which resulted in frameshift mutation. The wzc sequences of K9 and K45 exhibited high DNA sequence similarity but possessed different genes in their cps clusters. K52 and K79 exhibited 89% wzc DNA sequence identity but were readily distinguished from each other at the DNA level; in contrast, strains with the same capsular type as K52 exhibited 100% wzc sequence identity. A total of 29 strains from patients with bacteremia were typed by the wzc system. wzc DNA sequences confirmed the documented capsular type for twenty-eight of these clinical isolates; the remaining strain likely represents a new capsular type. Thus, the wzc genotyping system is a simple and useful method for capsular typing of K. pneumoniae.     

Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice

Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice.     

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: results of an in vitro and in vivo study

This study was designed to investigate the in vitro and in vivo transfection efficiency of chitosan nanoparticles used as vectors for gene therapy. Three types of chitosan nanoparticles [quaternized chitosan -60% trimethylated chitosan oligomer (TMCO-60%), C(43-45 KDa, 87%), and C(230 KDa, 90%)] were used to encapsulate plasmid DNA (pDNA) encoding green fluorescent protein (GFP) using the complex coacervation technique. The morphology, optimal chitosan-pDNA binding ratio and conditions for maximal in vitro transfection were studied. The in vivo transfection was conducted by feeding the chitosan/pDNA nanoparticles to 12 BALB/C-nu/nu nude mice. Both conventional and TMCO-60% could form stable nanoparticles with pDNA. The in vitro study showed the transfection efficiency to be in the following descending order: TMCO-60%>C(43-45 KDa, 87%)>C(230 KDa, 90%). TMCO-60% proved to be the most efficient and the optimal chitosan/pDNA ratio being 3.2:1. In vivo study showed most prominent GPF expression in the gastric and upper intestinal mucosa. GFP expression in the mucosa of the stomach and duodenum, jejunum, ileum, and large intestine were found, respectively, in 100%, 88.9%, 77.8% and 66.7% of the nude mice examined. TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two, and with minimal toxicity, which made it a desirable non-viral vector for gene therapy via oral administration.