Reversible (G0) and nonreadily reversible (Q) noncycling cells in human peripheral blood. Immunological, structural, and biological characterization

PHA-stimulated human lymphocytes (normal-resting-proliferating) at 0, 24, 48, 72, and 144 h were studied with Acridine Orange (AO) staining. By viable cell sorting, by subsequent subculturing, and by the use of biochemical, biophysical, and immunological assays, not only have the G0 resting and G1 (cycling) cell cycle phases been objectively characterized, but a separate subpopulation of quiescent cells that are functionally viable and deeply committed to nonproliferation, the Q cells, has been identified. Multiparameter cytofluorimetric analysis, methyl14C-thymidine incorporation, automated image analysis, and mitogen stimulation studies have shown that the "Q" cell, compared to the "G0" resting but easily recruitable cell, exhibits quite lower red and green AO emission, possesses 2c to 4c DNA content (rather than only 2c), has a higher average optical density, and is either nonrecruitable or recruitable-with-difficulty in PHA-stimulated lymphocyte cultures.     

Broadening the genetic base of European maize heterotic pools with US Cornbelt germplasm using field and molecular marker data

Maize (Zea mays L.) breeders are concerned about the narrowing of the genetic base of elite germplasm. To reverse this trend, elite germplasm from other geographic regions can be introgressed, but due to lack of adaptation it is difficult to assess their breeding potential in the targeted environment. The objectives of this study were to (1) investigate the relationship between European and US maize germplasm, (2) examine the suitability of different mega-environments and measures of performance to assess the breeding potential of exotics, and (3) study the relationship of genetic distance with mid-parent heterosis (MPH). Eight European inbreds from the Dent and Flint heterotic groups, 11 US inbreds belonging to Stiff Stalk (SS), non-Stiff Stalk (NSS), and CIMMYT Pool 41, and their 88 factorial crosses in F₁ and F₂ generations were evaluated for grain yield and dry matter concentration. The experiments were conducted in three mega-environments: Central Europe (target mega-environment), US Cornbelt (mega-environment where donor lines were developed), and Southeast Europe (an intermediate mega-environment). The inbreds were also fingerprinted with 266 SSR markers. Suitable criteria to identify promising exotic germplasm were F₁ hybrid performance in the targeted mega-environment and F₁ and parental performance in the intermediate mega-environment. Marker-based genetic distances reflected relatedness among the inbreds, but showed no association with MPH. Based on genetic distance, MPH, and F₁ performance, we suggest to introgress SS germplasm into European Dents and NSS into European Flints, in order to exploit the specific adaptation of European flint germplasm and the excellent combining ability of US germplasm in European maize breeding programs.     

Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates

Microglia are the resident macrophages in the central nervous system. In the spinal cord dorsal horn, microglia stay in resting condition during physiological sensory processing, and are activated under pathological conditions such as peripheral nerve injury. In cases such as this, the nearby resting microglia increase their motility and accumulate at the site of injury. However, direct evidence to support that nerve activity can enhance the motility of microglia has not yet to be reported. In this study we investigated whether the activation of spinal microglia under in vivo nerve injury may be mimicked by neuronal activity in the spinal cord slice preparation. We found that local application of spinal excitatory neurotransmitters, such as glutamate and substance P did not cause any change in the motility of microglial cells in the spinal cord dorsal horn. The motility of microglial cells is unlikely modulated by other transmitters, neuromodulators and chemokines, because similar applications such as GABA, serotonin, noradrenaline, carbachol, fractalkine or interleukin did not produce any obvious effect. Furthermore, low or high frequency stimulation of spinal dorsal root fibers at noxious intensities failed to cause any enhanced extension or retraction of the microglia processes. By contrast, focal application of ATP triggered rapid and robust activation of microglial cells in the spinal dorsal horn. Our results provide the first evidence that the activation of microglia in the spinal cord after nerve injury is unlikely due solely to neuronal activity, non-neuronal factors are likely responsible for the activation of nerve injury-related microglial cells in the spinal dorsal horn.     

A functional decaisoleucine-containing signal sequence. Construction by cassette mutagenesis

An alkaline phosphatase signal sequence optimized for formation of a hydrophobic alpha-helix functions very efficiently in the transport process. This mutant contained a core region comprised of 9 consecutive leucine residues (Kendall, D. A., Bock, S. C., and Kaiser, E. T. (1986) Nature 321, 706-708). We have now constructed a second mutant containing a decaisoleucine core region. Isoleucine was chosen because it is an isomer of leucine with comparable hydrophobicity but in synthetic peptides isoleucine favors beta-sheet formation. Surprisingly, this mutant precursor was also processed efficiently, and mature alkaline phosphatase was correctly targeted to the Escherichia coli periplasm. Since the effective length of a beta-strand is extended relative to an alpha-helix, conformational differences should be mirrored by the relative effectiveness of shortened polyisoleucine and polyleucine core regions. However, analysis of two additional mutants containing truncated segments of either polyleucine or polyisoleucine did not reveal any differences and both accumulate as precursors. We conclude that these mutants do not adopt critically different structures. This comparative analysis was facilitated by construction of a new plasmid, CASS3. This plasmid contains unique restriction sites flanking the DNA region coding for the signal sequence hydrophobic core segment. Consequently, the wild type core-encoding region can be readily replaced with synthetic oligonucleotides coding for new structural units and multiple amino acid substitutions can be made without the need for step-wise mutagenesis.