The relationship between the dominant Additional vein mutant in Drosophila melanogaster and engrailed.

Additional vein (Adv) is a dominant mutation that affects the first wing vein in Drosophila. It also manifests a recessive lethal phenotype and is associated with a large inversion. Using a combination of genetic and cytogenetic techniques, we show that Adv interacts with engrailed (en), likely because one of the inversion breakpoints interferes with en function. Genetic interaction studies reveal that Adv is lethal in trans with various lethal alleles of en and gives an engrailed-like wing phenotype with weak alleles of en. In situ hybridization to polytene chromosomes using en cDNA demonstrates that one of the inversion breakpoints lies within the en coding region. Although the cause of the wing phenotype is not determined herein, it likely is caused by the other inversion breakpoint interfering with a different function. The characterization of this mutation could expedite studies to understand what molecular events result in the Adv phenotype and thereby provide insight into the development of the first wing vein in Drosophila.     

Laser scanning cytometry in human brain slices.

BACKGROUND: The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections. METHODS: We adapted this technique to immunohistochemical labelled human brain slices. RESULTS: We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections. CONCLUSIONS: In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections.     

First-Year Common Nightingales (Luscinia megarhynchos) Have Smaller Song-Type Repertoire Sizes Than Older Males

Based on the assumptions that birdsong indicates male quality and that quality is related to age, one might expect older birds to signal their age. That is, in addition to actual body condition, at least some song features should vary with age, presumably towards more complexity. We investigated this issue by comparing repertoire sizes of free‐ranging common nightingale males in their first breeding season with those of older males. Nightingales are a good model species as they are open‐ended learners, where song acquisition is not confined to an early sensitive period of learning. Moreover, nightingales develop an extraordinarily large song‐type repertoire (approx. 180 different song types per male), and differences in repertoire size among males are pronounced. We analysed repertoire characteristics of the nocturnal song of nine nightingales in their first breeding season and compared them with the songs of nine older males. The repertoire size of older males was on average 53% larger than that of yearlings. When analysing two song categories of nightingales, whistle and non‐whistle songs separately, we found similar results. Our findings show marked differences in repertoire size between age categories, suggesting that this song feature may reflect a male's age. We discuss those mechanisms that may constrain the development of larger repertoires in first‐year males. Whether repertoire sizes are crucial for female mate choice or in vocal interactions among conspecific males remains open to further investigations.     

Produce Isolates of the Escherichia coli Ont:H52 Serotype That Carry both Shiga Toxin 1 and Stable Toxin Genes

Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group.     

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.     

Vaccine-Induced,Pseudorabies Virus-Specific,Extrathymic CD4+CD8+ Memory T-Helper Cells in Swine

Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4⁺CD8⁺ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4⁺CD8⁺ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4⁺CD8⁺ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4⁺CD8⁺ memory T lymphocytes and showed that CD4⁺CD8⁺ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4⁺CD8⁺ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, is the causative agent of Aujeszky’s disease. This disease is lethal to young pigs and causes important economic losses (52). Therefore, vaccination of pigs is practiced in many countries.Several humoral immune system effector mechanisms are involved in the protection of pigs from PRV infection. Virus-neutralizing antibodies, antibodies mediating antibody-dependent cell-mediated cytotoxicity, and antibodies mediating complement-mediated lysis of PRV-infected target cells have been demonstrated (22, 23, 53, 54). The main targets of this humoral immune response were shown to be the viral glycoproteins (3, 45), and passive immunization with monoclonal antibodies (MAbs) against gB, gC, and gD protects pigs from a lethal challenge (20, 49).The protection conferred through cell-mediated immunity is poorly understood. An increase in major histocompatibility complex (MHC)-unrestricted cell-mediated cytotoxicity against uninfected and PRV-infected cells has been detected after infection or vaccination of pigs with PRV (16, 53, 54), and specific cellular immune responses to PRV infections could be demonstrated by stimulation of proliferation and lymphokine secretion of porcine PRV-immune lymphocytes (10, 17, 42, 43, 51) as well as by the detection of PRV-specific cytotoxic lymphocytes (21, 56).There are some difficulties in defining more precisely the impact of cell-mediated immune effector mechanisms to protection from PRV-infection and their interplay with the observed humoral immune response. Considerably fewer porcine than human or mouse differentiation markers are available (34). In addition, the immune system of swine differs considerably from that of humans and mice. The pig has a substantial number of CD4⁻CD8⁻ T lymphocytes in the peripheral blood (4, 6, 12, 36, 39). In young animals, this subpopulation of T lymphocytes comprises up to 60% of the T lymphocytes and contains mainly γδ T lymphocytes. The pig is also the only species so far known to contain a substantial number of resting extrathymic CD4⁺CD8⁺ T lymphocytes (28, 36, 39). This T-lymphocyte population shows morphologically the phenotype of mature T lymphocytes (40) and increases with age to up to 60% of peripheral T lymphocytes (29, 35, 39, 55). Further, it was demonstrated that CD4⁺CD8⁺ T lymphocytes comprise memory T cells which proliferate upon stimulation with recall antigen (43, 55). Since the observed proliferative response was shown to be MHC class II-restricted, it was speculated that the porcine CD4⁺CD8⁺ T-cell subset contains memory T-helper lymphocytes (43). However, the ability of these T lymphocytes to secrete cytokines or to provide help to B cells has so far not been demonstrated.To gain a better understanding of immune effector mechanisms conferring protection from PRV infection, the function of these unusual extrathymic T-lymphocyte subsets has to be elucidated. In the present study, we identified two T-cell epitopes on glycoprotein gC which are primed during vaccination of d/d haplotype inbred pigs (41) against PRV and demonstrated that MHC class II-restricted, peripheral CD4⁺CD8⁺ memory T lymphocytes are the responding T lymphocytes. We were further able to show that PRV-specific, extrathymic CD4⁺CD8⁺ T lymphocytes are able to secrete cytokines and have the capacity to stimulate the secretion of PRV-specific immunoglobulins (Ig) by PRV-primed B cells. These results demonstrate that porcine CD4⁺CD8⁺ T lymphocytes can function as memory T-helper cells and can direct humoral anti-PRV memory responses.     

Storage behavior of Typha latifolia pollen at low water contents: Interpretation on the basis of water activity and glass concepts

Germplasm must be stored under optimal conditions to maximize longevity and efficiently maintain genetic resources. In order to identify optimal storage conditions, we investigated the effects of temperature (−5 to 45°C) and water content (<0.17 g H₂O g⁻¹ dry weight) on longevity of Typha latifolia L. pollen. Longevity was highest at water contents corresponding to storage relative humidity (RH) of 11‐15% which corresponded to the shoulder of water sorption isotherms. Also coinciding with this shoulder were abrupt changes in heat capacity of water present in the pollen. Consistent with changes in isotherms with temperature and the concept of critical RH for storage, optimum water contents increased with decreasing temperature. An attempt was made to explain the aging behavior according to the glass concept. The water content‐temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. We also found a change in activation energy of aging in Arrhenius plots around T_g, demonstrating a change in aging kinetics when the glassy state is lost. We concluL that T_g curves cannot be used solely to predict precise conditions of optimum storage, but might be useful for predictions of storage longevity above optimum water contents. The data imply that too much drying reduces longevity and should be avoided, particularly when cryogenic storage is considered.     

Human Brain Capillary Endothelium: Modulation of K+ Efflux and K+, Ca2+ Uptake by Endothelin

This report describes K⁺ efflux, K⁺ and Ca²⁺ uptake responses to endothelins (ET-1 and ET-3) in cultured endothelium derived from capillaries of human brain (HBEC). ET-1 dose dependently increased K⁺ efflux, K⁺ and Ca²⁺ uptake in these cells. ET-1 stimulated K⁺ efflux occurred prior to that of K⁺ uptake. ET-3 was ineffective. The main contributor to the ET-1 induced K⁺ uptake was ouabain but not bumetanide-sensitive (Na⁺-K⁺-ATPase and Na⁺-K⁺-Cl^– cotransport activity, respectively). All tested paradigms of ET-1 effects in HBEC were inhibited by selective antagonist of ET_A but not ET_B receptors and inhibitors of phospholipase C and receptor-operated Ca²⁺ channels. Activation of protein kinase C (PKC) decreased whereas inhibition of PKC increased the ET-1 stimulated K⁺ efflux, K⁺ and Ca²⁺ uptake in HBEC. The results indicate that ET-1 affects the HBEC ionic transport systems through activation of ET_A receptors linked to PLC and modulated by intracellular Ca²⁺ mobilization and PKC.