A Direct Screening Procedure for Gravitropism Mutants in Arabidopsis thaliana (L.) Heynh.

In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable.     

Analysis of genome-specific sequences inPhleum species: Identification and use for study of genomic relationships

Sau3AI shot gun cloning and colony hybridization with total genomic probes were used to isolate genome-specific sequences inPhleum species. The total DNA isolated from diploid speciesP. alpinum andP. bertolonii was partially digested withSau3AI and cloned using pUC19 as a vector to anE. coli strain DH5mcr. A partial genomic DNA library consisting of 3030 colonies for the genome ofP. alpinum and one consisting of 3240 colonies for the genome ofP. bertolonii were constructed. Twelve hundred and thirty colonies from the DNA library ofP. alpinum and 1320 from that ofP. bertolonii were respectively blotted to membrane filters and hybridized to the total genomic probes from these two species. Eight clones specific toP. alpinum and 13 specific toP. bertolonii were isolated through colony hybridization and further dot-blot hybridization. Most of these clones may carry highly or moderately repetitive sequences. Three sequences specific toP. alpinum and 3 specific toP. bertolonii were used as probes to hybridize theEcoRI-digested DNA samples from four species,P. alpinum,P. bertolonii,P. pratense andP. montanum, on Southern blot. The results from these hybridization experiments showed that all 3P. bertolonii-specific probes and 2 of the 3P. alpinum-specific probes hybridized to the DNA ofP. pratense, thus confirming the conclusion of the close relationships between the cultivated timothy and its two wild relatives that was drawn in our previous study using the C-banding technique.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Anaerobic CO2 Cabinet for the Cultivation of Strict Anaerobes

The design and construction of an anaerobic CO(2) cabinet are described. Air is displaced by a stream of oxygen-free CO(2), and anerobic conditions are produced in 3 hr. The equipment is simple and cheap to operate and has been found to be satisfactory for the isolation of strict anaerobes from the mouse intestine.     

Effect of photosynthetic biofilms on the open‐circuit potential of stainless steel

Periodic illumination of photosynthetic biofilms on AISI* 316L stainless steel resulted in evolution of oxygen (1–7 mg.1^‐1) and a corresponding increase in open circuit potential (E_corr) from 2 to 15 mV. The change in E^ depended on the interval of illumination. When the dark cycle began, elevation in potential was followed by an immediate drop. Illumination did not affect E_corr in sterile systems or in systems that contained only nonphotosynthetic eubacteria. Radiated heat from illumination accounted for changes of 4 to 5°C in temperature which, in the absence of oxygen production, should decrease dissolved oxygen by 0.75 mgl^‐1 and decrease E_corr by 1 mV. Positive shifts of E_corr induced by periodic illumination of photosynthetic biofilms are primarily the result of oxygen production.     

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.     

Short-term resistance to diet-induced obesity in A/J mice is not associated with regulation of hypothalamic neuropeptides

To investigate the mechanisms underlying long-term resistance of the A/J mouse strain to diet-induced obesity, we studied, over a period of 4 wk, the expression of uncoupling proteins in brown adipose tissue and the expression of hypothalamic neuropeptides known to regulate energy homeostasis and then used microarray analysis to identify other potentially important hypothalamic peptides. Despite increased caloric intake after 2 days of high-fat feeding, body weights of A/J mice remained stable. On and after 1 wk of high-fat feeding, A/J mice adjusted their food intake to consume the same amount of calories as mice fed a low-fat diet; thus their body weight and insulin, corticosterone, free fatty acid, and glucose levels remained unchanged for 4 wk. We found no changes in hypothalamic expression of several orexigenic and/or anorexigenic neuropeptides known to play an important role in energy homeostasis for the duration of the study. Uncoupling protein-2 mRNA expression in brown adipose tissue, however, was significantly upregulated after 2 days of high-fat feeding and tended to remain elevated for the duration of the 4-wk study. Gene array analysis revealed that several genes are up- or downregulated in response to 2 days and 1 wk of high-fat feeding. Real-time PCR analysis confirmed that expression of the hypothalamic IL-1 pathway (IL-1beta, IL-1 type 1 and 2 receptors, and PPM1b/PP2C-beta, a molecule that has been implicated in the inhibition of transforming growth factor-beta-activated kinase-1-mediated IL-1 action) is altered after 2 days, but not 1 wk, of high-fat feeding. The role of additional molecules discovered by microarray analysis needs to be further explored in the future.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.