Marker-assisted selection for white mold resistance in common bean

A marker-assisted selection (MAS) study was conducted on two recombinant inbred line (RIL) populations of common bean (Phaseolus vulgaris) to test the effectiveness of MAS for resistance to white mold (Sclerotinia sclerotiorum). Markers for quantitative trait loci (QTL) on linkage groups B2 and B7 that were previously associated with resistance and plant architectural avoidance traits in the resistant parent Bunsi were chosen. In the Bunsi/Midland population 10 RILs included in the MAS selected group developed significantly less disease than the control group based on two years of field evaluation under white mold pressure. Growth habit had no significant effect on disease severity or incidence. In the Bunsi/Raven RIL population, disease scores in the MAS selected group were significantly lower than scores in the control group over two years. Additional progress in enhancing resistance to white mold was detected when yield and plant architecture were included in the selection process. Lower disease scores among RILs were observed when comparisons were made to RILs selected using MAS alone. Yield is an important trait that should be considered when selecting for resistance to white mold. Finally the potential of Bunsi as a genetic donor of QTL for white mold resistance was confirmed in both populations studied. This study supported the effectiveness of MAS to enhance selection for a complexly inherited trait such as resistance to white mold in common bean.     

High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [¹³C₁₈]oleic acid. The [¹³C₁₈]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [¹³C₁₈]oleic acid to rats. The [¹³C₁₈]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [¹³C₁₈]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [¹³C₁₈]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [¹³C₁₈]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.     

MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.     

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10^?8M and was found to be completely blocked by indomethacin. Adding back 10^?8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.     

Two forms of glutamine synthetase in free-living root-nodule bacteria.

Cell-free extracts of $\text{Rhizobium japonicum}$ 61A76 contain two forms of glutamine synthetase (EC 6.3.1.2) which can be easily separated by isoelectric focusing. The more acid form (pI = 5.4), like the enzyme from $\text{E. coli}$, is stable at 50° and catalyses an ADP-dependent transferase reaction, whose inhibition by excess Mg⁺⁺ can be relieved by snake venom phosphodiesterase. The more alkaline form (pI = 6.1) is labile at 50°, and catalyses and ADP-dependent transferase reaction which is strongly inhibited by Mg⁺⁺ regardless of phosphodiesterase treatment. We have also observed the two forms of the enzyme in a nitrogenaseless mutant of 61A76, and in other strains of rhizobia, but only the acid form in $\text{E. coli}$ W, $\text{A. vinelandii}$ OP, and $\text{K. pneumoniae}$ M 51A.     

Electron tomographic and ultrastructural analysis of the Cryptosporidium parvum relict mitochondrion, its associated membranes, and organelles

Sporozoites of the apicomplexan Cryptosporidium parvum possess a small, membranous organelle sandwiched between the nucleus and crystalloid body. Based upon immunolabelling data, this organelle was identified as a relict mitochondrion. Transmission electron microscopy and tomographic reconstruction reveal the complex arrangement of membranes in the vicinity of this organelle, as well as its internal organization. The mitochondrion is enveloped by multiple segments of rough endoplasmic reticulum that extend from the outer nuclear envelope. In tomographic reconstructions of the mitochondrion, there is either a single, highly-folded inner membrane or multiple internal subcompartments (which might merge outside the reconstructed volume). The infoldings of the inner membrane lack the tubular "crista junctions" found in typical metazoan, fungal, and protist mitochondria. The absence of this highly conserved structural feature is congruent with the loss, through reductive evolution, of the normal oxidative phosphorylation machinery in C. parvum. It is proposed that the retention of a relict mitochondrion in C. parvum is a strategy for compartmentalizing away from the cytosol toxic ferrous iron and sulfide, which are needed for iron sulfur cluster biosynthesis, an essential function of mitochondria in all eukaryotes.     

Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.     

Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms of Chromosome Congression and Anaphase Onset

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK₁ cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late metaphase 24.3 ± 4.9 (n = 62), and at early anaphase 27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 μM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.     

Synthetic Orcein as an Elastic Tissue Stain

A study was made of various synthetic orceins in an effort to determine the optimal conditions for their use in staining elastic tissue. A simple technic has been developed, based on a modification of the method originally worked out by Taenzer. Orcein is used as a 0.4% solution in 70% alcohol containing 1% HC1. Sections are counterstained with Mallory's borax methylene blue. The solutions are easily prepared and may be used for several months. Although several methods require staining in orcein from 2 to 24 hours, the present method requires staining for only 30 minutes. After several types of fixation some of the dye samples stained collagenous tissue to varying degrees, but this could be diminished by slightly reducing the strength of the staining solution. Differential staining of elastic tissue was particularly specific in tissues fixed in acetone, collagen in these preparations remaining practically unstained by any of the dye samples.