Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations

Divalent cations are thought essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the effects of 10 mM EDTA on locomotion of human blood polymorphonuclear leukocytes (PMN). In very thin slide preparations, EDTA did not impair either random locomotion or chemotaxis; motile behavior appeared to benefit from the close approximation of slide and coverslip (“chimneying”). In preparations twice as thick, PMN in EDTA first exhibited active deformability with little or no displacement, then rounded up and became motionless. However, on creation of a chemotactic gradient, the same cells were able to orient and make their way to the target, often, however, losing momentarily their purchase on the substrate. In either of these preparations without EDTA, specific antibodies to β2 integrins did not prevent random locomotion or chemotaxis, even when we added antibodies to β1 and αvβ3 integrins and to integrin-associated protein, and none of these antibodies added anything to the effects of EDTA. In the more turbulent environment of even more media, effects of anti-β2 integrins became evident: PMN still could locomote but adhered to substrate largely by their uropods and by uropod-associated filaments. We relate these findings to the reported independence from integrins of PMN in certain experimental and disease states. Moreover, we suggest that PMN locomotion in close quarters is not only integrin-independent, but independent of external divalent cations as well.

Miriamin, a defensive diterpene from the eggs of a land slug (Arion sp.)

The eggs of the land slug Arion sp. contain a diterpene, miriamin, characterized as a polyoxygenated geranylgeraniol derivative. In bioassays with a coccinellid beetle, Harmonia axyridis, miriamin was shown to be potently antifeedant, indicating that the compound plays a protective role in nature. It is suggested that mucilaginous soil-inhabiting organisms, given their intense exposure to pathogens and predators, may be a rich source of chemical defensive agents.

Silencer elements controlling the B29 (Igβ) promoter are neither promoter- nor cell-type-specific

The murine B29 (Igβ) promoter is B cell specific and contains essential SP1, ETS, OCT, and Ikaros motifs. Flanking 5′ DNA sequences inhibit B29 promoter activity, suggesting this region contains silencer elements. Two adjacent 5′ DNA segments repress transcription by the murine B29 promoter in a position- and orientation-independent manner, analogous to known silencers. Both these 5′ segments also inhibit transcription by several heterologous promoters in B cells, including mb-1, c-fos, and human B29. These 5′ segments also inhibit transcription by the c-fos promoter in T cells suggesting they are not B cell-specific elements. DNase I footprint analyses show an approximately 70-bp protected region overlapping the boundary between the two negative regulatory DNA segments and corresponding to binding sites for at least two different DNA-binding proteins. Within this footprint, two unrelated 30-bp cis-acting DNA motifs (designated TOAD and FROG) function as position- and orientation-independent silencers when located directly 5′ of the murine B29 promoter. These two silencer motifs act cooperatively to restrict the transcriptional activity of the B29 promoter. Neither of these motifs resembles any known silencers. Mutagenesis of the TOAD and FROG motifs in their respective 5′ DNA segments eliminates the silencing activity of these upstream regions, indicating these two motifs as the principal B29 silencer elements within these regions.

Regulation of Oleoresinosis in Grand Fir (Abies grandis)1 : Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding

Grand fir (Abies grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.