Integration of the cytogenetic and genetic linkage maps of Brassica oleracea

We have assigned all nine linkage groups of a Brassica oleracea genetic map to each of the nine chromosomes of the karyotype derived from mitotic metaphase spreads of the B. oleracea var. alboglabra line A12DHd using FISH. The majority of probes were BACs, with A12DHd DNA inserts, which give clear, reliable FISH signals. We have added nine markers to the existing integrated linkage map, distributed over six linkage groups. BACs were definitively assigned to linkage map positions through development of locus-specific PCR assays. Integration of the cytogenetic and genetic linkage maps was achieved with 22 probes representing 19 loci. Four chromosomes (2, 4, 7, and 9) are in the same orientation as their respective linkage groups (O4, O7, O8, and O6) whereas four chromosomes (1, 3, 5, and 8) and linkage groups (O3, O9, O2, and O1) are in the opposite orientation. The remaining chromosome (6) is probably in the opposite orientation. The cytogenetic map is an important resource for locating probes with unknown genetic map positions and is also being used to analyze the relationships between genetic and cytogenetic maps.     

Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity

The epidermal Langerhans cells (LC), a member of the dendritic cell family, and the LC-derived cytokine IL-12 play a pivotal role in the initiation of contact hypersensitivity (CHS), a Th1 immune response in the skin. Because IL-18, another LC-derived cytokine, shares functional and biological properties with IL-12, we examined a potential role for IL-18 in CHS initiation. Our studies demonstrated that during the induction phase of murine CHS, IL-18 mRNA was significantly up-regulated in the skin-draining lymph nodes (LN). Migratory hapten-modified LC in LN expressed high levels of IL-18 mRNA and secreted functional IL-18 protein. LN cells produced significant amounts of IFN-gamma following in vitro IL-12 stimulation, which could be partially blocked by anti-IL-18 Ab, suggesting a synergistic role for endogenous IL-18 in IFN-gamma production by LN cells. Because mature IL-18 requires cleavage of immature precursors by caspase-1, we further examined IL-12-induced IFN-gamma production in caspase-1(-/-) LN cells. An impaired IFN-gamma production was seen in caspase-1(-/-) LN cells, which could be restored by addition of exogenous IL-18, supporting a role for caspase-1-cleaved, mature IL-18 in IFN-gamma production. Finally, in vivo studies showed that CHS responses were significantly inhibited in mice treated with neutralizing IL-18 Ab as well as in caspase-1(-/-) mice deficient in mature IL-18, indicating functional relevance for IL-18 in CHS. Taken together, our studies demonstrate that LC-derived IL-18 significantly contributes to CHS initiation.     

Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups

The evolution of the human mitochondrial genome is characterized by the emergence of ethnically distinct lineages or haplogroups. Nine European, seven Asian (including Native American), and three African mitochondrial DNA (mtDNA) haplogroups have been identified previously on the basis of the presence or absence of a relatively small number of restriction-enzyme recognition sites or on the basis of nucleotide sequences of the D-loop region. We have used reduced-median-network approaches to analyze 560 complete European, Asian, and African mtDNA coding-region sequences from unrelated individuals to develop a more complete understanding of sequence diversity both within and between haplogroups. A total of 497 haplogroup-associated polymorphisms were identified, 323 (65%) of which were associated with one haplogroup and 174 (35%) of which were associated with two or more haplogroups. Approximately one-half of these polymorphisms are reported for the first time here. Our results confirm and substantially extend the phylogenetic relationships among mitochondrial genomes described elsewhere from the major human ethnic groups. Another important result is that there were numerous instances both of parallel mutations at the same site and of reversion (i.e., homoplasy). It is likely that homoplasy in the coding region will confound evolutionary analysis of small sequence sets. By a linkage-disequilibrium approach, additional evidence for the absence of human mtDNA recombination is presented here.     

Structure of the Golgi and distribution of reporter molecules at 20 degrees C reveals the complexity of the exit compartments

Incubating cells at 20 degrees C blocks transport out of the Golgi complex and amplifies the exit compartments. We have used the 20 degrees C block, followed by EM tomography and serial section reconstruction, to study the structure of Golgi exit sites in NRK cells. The dominant feature of Golgi structure in temperature-blocked cells is the presence of large bulging domains on the three trans-most cisternae. These domains extend laterally from the stack and are continuous with "cisternal" domains that maintain normal thickness and alignment with the other stacked Golgi cisternae. The bulging domains do not resemble the perpendicularly extending tubules associated with the trans-cisternae of control cells. Such tubules are completely absent in temperature-blocked cells. The three cisternae with bulging domains can be identified as trans by their association with specialized ER and the presence of clathrin-coated buds on the trans-most cisterna only. Immunogold labeling and immunoblots show a significant degradation of a medial- and a trans-Golgi marker with no evidence for their redistribution within the Golgi or to other organelles. These data suggest that exit from the Golgi occurs directly from three trans-cisternae and that specialized ER plays a significant role in trans-Golgi function.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on phospholipid monolayers. Protein structure studied using 2D IR and beta correlation analysis

We have applied two-dimensional infrared (2D IR) and betanu correlation spectroscopy to in-situ IR spectroscopy of pulmonary surfactant proteins SP-B and SP-C in lipid-protein monolayers at the air-water interface. For both SP-B and SP-C, a statistical windowed autocorrelation method identified two separate surface pressure regions that contained maximum amide I intensity changes: 4-25 mN/m and 25-40 mN/m. For SP-C, 2D IR and betanu correlation analyses of these regions indicated that SP-C adopts a variety of secondary structure conformations, including alpha-helix, beta-sheet, and an intermolecular aggregation of extended beta-sheet structure. The main alpha-helix band split into two peaks at high surface pressures, indicative of two different helix conformations. At low surface pressures, all conformations of the SP-C molecule reacted identically to increasing surface pressure and reoriented in phase with each other. Above 25 mN/m, however, the increasing surface pressure selectively affected the coexisting protein conformations, leading to an independent reorientation of the protein conformations. The asynchronous 2D IR spectrum of SP-B showed the presence of two alpha-helix components, consistent with two separate populations of alpha-helix in SP-B-a hydrophobic fraction associated with the lipid chains and a hydrophilic fraction parallel to the membrane surface. The distribution of correlation intensity between the two alpha-helix cross peaks indicated that the more hydrophobic helix fraction predominates at low surface pressures whereas the more hydrophilic helix fraction predominates at high surface pressures. The different SP-B secondary structures reacted identically to increasing surface pressure, leading to a reorientation of all SP-B subunits in phase with one another.     

Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.     

Effect of active vs. passive recovery on repeat suicide run time

This study was conducted to evaluate the difference between active and passive recovery methods during successive suicide runs by Division I women's collegiate basketball athletes (n = 14). Testing consisted of sprinting suicides on the basketball court using both traditional (short) and reverse-sequence (long) protocols. Two 90-second recovery methods were used, passive (standing still) and active (slow self-paced jogging). Although successive run time was reduced by a mean of 0.55 seconds after passive recovery relative to active, it did not reach significance (p = 0.09). Likewise, the difference between long and short line versions was nonsignificant (p = 0.41). Therefore, neither line sequence nor 90-second recovery technique appears to influence subsequent run time when performing 2 maximal-effort suicides.     

A method for the comprehensive proteomic analysis of membrane proteins

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.