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Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.

Background Cardiomyocytes are resistant to radiation. However, cardiac radiation exposure causes coronary microvascular endothelial inflammation, a perturbation implicated in the pathogenesis of heart failure (HF) and particularly, HF with preserved ejection fraction (HFpEF). Radiotherapy for breast cancer results in variable cardiac radiation exposure and may increase the risk of HF. Methods We conducted a population-based case-control study of incident HF in 170 female residents of Olmsted County, Minnesota (59 cases and 111 controls) who underwent contemporary (1998–2013) radiotherapy for breast cancer utilizing computed tomography-assisted radiotherapy planning. Controls were matched to cases for age, tumor side, chemotherapy use, diabetes and hypertension. Mean cardiac radiation dose (MCRD) in each patient was calculated from their computed tomography images and radiotherapy plan. Results Mean age at radiotherapy was 69±9 years. Of HF cases, 38 (64%) had EF≥ 50% (HFpEF), 18 (31%) had EF<50% (HFrEF) and 3 (5%) did not have EF measured. The EF was ≥ 40% in 50 (89%) of the 56 HF cases with an EF measurement. The mean interval from radiotherapy to HF was 5.8±3.4 years. The odds of HF was higher in patients with a prior history of ischemic heart disease or atrial fibrillation. The MCRD was 2.5 Gy (range 0.2 to 13.1 Gy) and higher in cases (3.3±2.7 Gy) than controls (2.1±2.0 Gy, p=0.004). The odds ratio (95% confidence interval) for HF per log MCRD was 9.1 (3.4, 24.4) for any HF, 16.9 (3.9,73.7) for HFpEF and 3.17 (0.8,13.0) for HFrEF. The increased odds of any HF or HFpEF with increasing MCRD remained significant after adjustment for HF risk factors and in sensitivity analyses matching by cancer stage rather than tumor side. Only 18.6% of patients experienced new or recurrent ischemic events between radiotherapy and onset of HF. Conclusion The relative risk of HFpEF increases with increasing cardiac radiation exposure during contemporary conformal breast cancer radiotherapy. These data emphasize the importance of radiotherapy techniques which limit MCRD during breast cancer treatment. Moreover, these data provide further support for the importance of coronary microvascular compromise in the pathophysiology of HFpEF.

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated antispecies secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical limits of detectability. The optimum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd of the antibody-estriol interaction, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody.

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has been known as an aerobe that respires on iron and sulfur. Here we show that the bacterium could chemolithoautotrophically grow not only on H 2 /O 2 under aerobic conditions but also on H 2 /Fe 3؉ , H 2 /S 0 , or S 0 /Fe 3؉ under anaerobic conditions. Anaerobic respiration using Fe 3؉ or S 0 as an electron acceptor and H 2 or S 0 as an electron donor serves as a primary energy source of the bacterium. Anaerobic respiration based on reduction of Fe 3؉ induced the bacterium to synthesize significant amounts of a c-type cytochrome that was purified as an acid-stable and soluble 28-kDa monomer. The purified cytochrome in the oxidized form was reduced in the presence of the crude extract, and the reduced cytochrome was reoxidized by Fe 3؉ . Respiration based on reduction of Fe 3؉ coupled to oxidation of a c-type cytochrome may be involved in the primary mechanism of energy production in the bacterium on anaerobic iron respiration.Acidithiobacillus ferrooxidans is generally accepted to be an aerobic chemolithoautotroph that derives energy for growth from oxidative respiration involving the oxidation of ferrous iron or various sulfur compounds. Brock and Gustafson reported that the bacterium reduces Fe 3ϩ in the presence of S 0 (10). However, the reduction was not recognized as respiratory reactions since iron reduction did not permit growth of the bacterium (39). Therefore, it was accepted that coupling reduction of Fe 3ϩ to oxidation of S 0 was one of the steps in the sulfur metabolism by the bacterium (39). Pronk et al. (32,33) and Das et al. (12) showed that the bacterium grew on the oxidation of S 0 by Fe 3ϩ under oxygen-limited conditions. Although these findings raised the possibility that A. ferrooxidans might be able to grow under anaerobic conditions, the aforementioned enzymatic activity was not accompanied by growth (39), and it is still unclear whether Fe 3ϩ serves as an electron acceptor for anaerobic respiration. On the other hand, the bacterium would grow on hydrogen under aerobic conditions (14). In that case, H 2 served as the electron donor enabling an oxidative respiratory chain to derive energy for chemolithoautotrophic growth.On the other hand, in many facultative heterotrophs in both Archaea and Bacteria, anaerobic respiration involving reduction of Fe 3ϩ or S 0 is typically coupled to the oxidation of H 2 (25,26,30,38,42). One of the principle roles of such respiration would have been to support energy for chemolithoautotrophy (34), a type of autotrophy that typically served as the growth mode of such facultative heterotrophs in Archaea and Bacteria as hyperthermophilic archaebacteria, sulfur-reducing bacteria, and primitive fermentative bacteria (18, 24, 36-38, 42, 44). However, little is known about the role played by anaerobic respiration involving Fe 3ϩ or S 0 reduction in the growth of typical, known chemolithoautotrophic bacteria, which include a variety of sulfur, iron, ammonia, and nitrite oxidizers. We have found tha...

The mechanism of peroxisome proliferation is poorly understood. Candida boidinii is a methylotrophic yeast that undergoes rapid and massive peroxisome proliferation and serves as a good model system for this process. Pmp30A and Pmp30B (formerly designated Pmp31 and Pmp32, respectively) are two closely related proteins in a polyploid strain of this yeast that are strongly induced by diverse peroxisome proliferators such as methanol, oleate, and D-alanine. The function of these proteins is not understood. To study this issue, we used a recently described haploid strain (S2) of C. boidinii that can be manipulated genetically. We now report that strain S2 contains a single PMP30 gene very similar in sequence (greater than 93% identity at the DNA level) to PMP30A and PMP30B. When PMP30 was disrupted, cell growth on methanol was greatly inhibited, and cells grown in both methanol and oleate had fewer, larger, and more spherical peroxisomes than wild-type cells. A similar phenotype was recently described for Saccharomyces cerevisiae cultured on oleate in which PMP27, which encodes a protein of related sequence that is important for peroxisome proliferation, was disrupted. To determine whether Pmp27 is a functional homolog of Pmp30, genetic complementation was performed. PMP30A was expressed in the PMP27 disruptant of S. cerevisiae, and PMP27 was expressed in the PMP30 disruptant of C. boidinii S2. Complementation, in terms of both cell growth and organelle size, shape, and number, was successful in both directions, although reversion to a wild-type phenotype was only partial for the PMP30 disruptant. We conclude that these proteins are functional homologs and that both Pmp30 and Pmp27 have a direct role in proliferation and organelle size rather than a role in a specific peroxisomal metabolic pathway of substrate utilization.

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Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome- deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.