Significant collections of Orchidaceae conserved in Herbarium Hamburgense (HBG)

An inventory of critical type material in the recently rediscovered Kraenzlin Orchid Herbarium is presented. In addition, a partial inventory of type material of Orchidaceae conserved in the general herbarium at Hamburg (HBG) is presented as a guide to the collections.     

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo.In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.     

Detergent-activated BAX Protein Is a Monomer

BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation with detergents, such as n-octyl glucoside. During BAX activation with n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.BAX² is a pro-apoptotic member of the BCL-2 protein family. In a simplified apoptosis model, monomeric inactive BAX is localized in the cytoplasm of healthy nondying cells (1). During apoptosis BAX is activated and translocates to the outer mitochondrial membrane (2) where it inserts as a monomer (3), undergoes oligomerization (4), and forms a pore through which cytochrome c and other apoptotic factors are released into the cytoplasm. Once in the cytoplasm, these apoptotic factors induce the activation of the effector caspases that execute the cell death process. This mechanism, which is generally correct, requires that soluble BAX becomes integrated into the mitochondrial membrane where it forms a functional oligomeric pore capable of cytochrome c release. However, the molecular mechanism of BAX activation remains controversial (5, 6).It has been understood for some time, but frequently ignored, that activity of the BCL-2 family proteins is exhibited in cells when these proteins are associated with the hydrophobic environment of membranes. Therefore, it has always seemed that attention to the effect of hydrophobic environments on the BCL-2 family proteins would be rewarding. It has been shown that BAX can be directly activated by treatment with nonionic detergents such as n-octyl glucoside, dodecyl maltoside, and Triton X-100 (1, 7). During activation by nonionic detergents, to gain the ability to form pores in a bilayer membrane, BAX needs to undergo a major conformational transition from a globular protein with two pore-forming α-helices 5 and 6 hidden in the protein core (8) to a conformation in which these two helices are exposed and inserted into a lipid membrane (3, 5, 9). The nature of this active conformation of BAX is important for the understanding of the death decision in cells. Most proposals suggest that in a cell this activated form of BAX protein is initiated and maintained by the interactions with other proteins, such as tBID, or by BAX itself as a homo-oligomer (7, 10).Nonionic detergents have been commonly used to activate BAX for in vitro studies because they are reliably effective and simple to employ. However, little is known about the detailed molecular mechanism of BAX activation by these detergents and its comparability with in vivo activation of BAX. What is known is that concentrations of detergent above their critical micelle concentration (CMC) are necessary for BAX activation. This suggests that, to be activated, BAX needs to interact with detergent micelles instead of monomeric detergent molecules. For example, in the case of BAX activation by n-octyl glucoside, it has been shown that n-octyl glucoside concentration should be 1% (w/v) (7), which is well above the CMC for this detergent (0.6% w/v) (11). In addition, it has also been shown that above their individual CMC concentrations most BAX-activating detergents produce a change in BAX conformation that can be detected by a conformation-sensitive 6A7 antibody against BAX (1, 12, 13). In cellular experiments this feature of BAX reactivity to 6A7 antibody is commonly associated with the onset of apoptosis (14, 15). However, CHAPS does not generate the antibody-detected conformational change or the activation of BAX. The small micelle size of this detergent (10 kDa) suggests that perhaps BAX cannot adopt an activated state with this detergent. However, cholic acid with even smaller micelle size (4 kDa) can partially activate BAX (1).Many important detergent properties are associated with micelles. The formation of detergent micelles in solution is concentration-dependent beginning at the CMC. The CMC value for a detergent has practical importance because in most cases only monomers of detergent can be removed by dialysis, and therefore, it is easier to remove detergent monomers for a detergent with high CMC value than for a detergent with low CMC (11). For BAX this same consideration applies to its activation with n-octyl glucoside (CMC ∼23 mm) as compared with its activation with Triton X-100 (CMC ∼0.25 mm). The ease of dialysis is why, in most cases, OG is used to activate BAX in vitro.It has been shown by analytical gel filtration that, when incubated with n-octyl glucoside, BAX creates complexes with molecular weight larger than the combined size of a BAX monomer (21 kDa) and an n-octyl glucoside micelle (∼26 kDa) (7, 11). It has also been shown that in defined liposomes BAX pore formation requires oligomerization (16). These data combined with the knowledge that oligomerization is important for the biological function of BAX led to a hypothesis that BAX oligomerizes during its detergent activation prior to membrane insertion (7). However, it has been shown that in vivo activated BAX inserts into the outer mitochondrial membrane as a monomer (3), and to create a pore, BAX undergoes oligomerization in this membrane (4). This discrepancy between the oligomeric state of active BAX prior to its insertion into a lipid membrane in vivo (monomer) and in vitro (possibly hexamer or octamer) led us to study the oligomerization state of BAX in detergent micelles. The important issue is whether BAX activation requires protein oligomerization or whether active BAX conformation can be generated from a single protein monomer. To solve this issue we used two single-molecule sensitivity techniques: fluorescence correlation spectroscopy (FCS) (17) and fluorescence-intensity distribution analysis (FIDA) (18). Combined use of FCS and FIDA allows simultaneous determination of the apparent molecular weight and the number of fluorescently labeled BAX monomers per protein-detergent micelle. Our results are consistent with previously established results in which BAX forms high molecular weight protein-detergent micelles with n-octyl glucoside (4) and show that BAX is present as a monomer in these complexes. In addition, we determined the apparent molecular weight and the number of BAX proteins bound per protein-detergent micelles formed by BAX and micelles of five additional detergents (dodecyl maltoside, Triton X-100, Tween 20, cholic acid, and CHAPS). Our data show that BAX is a monomer before, during, and after interaction with the micelles of all tested detergents.     

Micropropagation by tissue culture triggers differential expression of infectious endogenous Banana streak virus sequences (eBSV) present in the B genome of natural and synthetic interspecific banana plantains

The genome of Musa balbisiana spp. contains several infectious endogenous sequences of Banana streak virus (eBSV). We have shown previously that in vitro micropropagation triggers the activation of infectious eBSOLV (endogenous sequences of Banana streak Obino l'Ewai virus ) in the synthetic tetraploid interspecific hybrid FHIA21 (AAAB). In this work, we show that another synthetic tetraploid (AAAB) hybrid and two natural triploid (AAB) plantains are equally prone to the activation of infectious eBSOLV during tissue culture. These results are a strong indication that such activation is a general phenomenon in interspecific Musa cultivars, whether synthetic or natural. We also report the first in-depth study of the correlation between the duration of tissue culture and the level of activation of infectious eBSOLV, and show that specific and common activation patterns exist in these banana plants. We hypothesize that these patterns result from the concomitant activation of infectious eBSOLV and a decrease in the virus titre in neoformed plantlets, resulting from cell multiplication outcompeting virus replication. We provide experimental data supporting this hypothesis. No activation of infectious eBSGFV (endogenous sequences of Banana streak Goldfinger virus) by tissue culture was observed in the two natural AAB plantain cultivars studied here, whereas such activation occurred in the AAAB synthetic hybrid studied. We demonstrate that this differential activation does not result from differences in the structure of eBSGFV, as all banana genomes harbour eaBSGFV-7.     

Effects of moderate ammonium enrichment on three submersed macrophytes under contrasting light availability

1. Increased ammonium concentrations and decreased light availability in a water column have been reported to adversely affect submersed vegetation in eutrophic waters worldwide. 2. We studied the chronic effects of moderate enrichment (NH₄–N: 0.16–0.25 mg L^?1) on the growth and carbon and nitrogen metabolism of three macrophytes (Ceratophyllum demersum, Myriophyllum spicatum and Vallisneria natans) under contrasting light availability in a 2‐month experiment. 3. The enrichment greatly increased the contents of free amino acids and nitrogen in the shoot / leaf of the macrophytes. This indicates that was the dominant N source for the macrophytes. 4. Soluble carbohydrate contents remained relatively stable in the shoot / leaf of the macrophytes irrespective of the treatments. Under ambient light, the starch contents in the shoot / leaf of C. demersum and M. spicatum increased with enrichment, whereas V. natans did not exhibit any change. The starch contents decreased in C. demersum, increased in M. spicatum and remained unchanged in V. natans after the combined treatment of enrichment and reduced light. 5. The enrichment did not affect the growth of the three macrophytes under the ambient light. However, it did suppress the growth of C. demersum and M. spicatum under the reduced light. The results indicate that a moderate enrichment was not directly toxic to the macrophytes although it might change their viability in eutrophic lakes in terms of the carbon and nitrogen metabolism.     

Winter climate change affects growing‐season soil microbial biomass and activity in northern hardwood forests

Understanding the responses of terrestrial ecosystems to global change remains a major challenge of ecological research. We exploited a natural elevation gradient in a northern hardwood forest to determine how reductions in snow accumulation, expected with climate change, directly affect dynamics of soil winter frost, and indirectly soil microbial biomass and activity during the growing season. Soils from lower elevation plots, which accumulated less snow and experienced more soil temperature variability during the winter (and likely more freeze/thaw events), had less extractable inorganic nitrogen (N), lower rates of microbial N production via potential net N mineralization and nitrification, and higher potential microbial respiration during the growing season. Potential nitrate production rates during the growing season were particularly sensitive to changes in winter snow pack accumulation and winter soil temperature variability, especially in spring. Effects of elevation and winter conditions on N transformation rates differed from those on potential microbial respiration, suggesting that N‐related processes might respond differently to winter climate change in northern hardwood forests than C‐related processes.     

Effect of follicular fluid treatment on follicle-stimulating hormone, luteinizing hormone and compensatory ovarian hypertrophy in prepuberal gilts

Prepuberal 130-day-old gilts were treated with 10 ml of charcoal-stripped porcine serum (PS), whole porcine follicular fluid (WpFF) or charcoal-stripped pFF (CpFF) twice daily beginning the day before and continuing 8 days after unilateral ovariectomy (ULO). Follicle-stimulating hormone (FSH) declined for the first 14 h after ULO in WpFF and CpFF gilts and then by 24 h returned to values observed at or before ULO, whereas FSH was increased nearly twofold at 14 h in PS gilts. At 8 days after ULO the remaining ovaries from PS-treated gilts were heavier than ovaries from follicular fluid-treated gilts. In a second experiment, ovariectomized 130-day-old gilts were assigned to either a group infused with PS, a group infused with 5 ml CpFF, or a group infused with 10 ml Cpff at 18 and 2 h before a gonadotropin-releasing hormone (GnRH) challenge. Porcine follicular fluid had no effect on luteinizing hormone (LH) response to GnRH, depressed the FSH response to a 10-micrograms challenge of GnRH, but had no effect on FSH response to a 50-micrograms challenge of GnRH. In a third study, gilts were subjected to sham ovariectomy (Sham) or ULO at 130 days of age. GnRH (10 micrograms) was given on Days 1, 2 or 8 after surgery. The response to GnRH in ULO versus Sham gilts did not differ for FSH or LH on any day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic clearance and production rates of oestradiol and progesterone during pubertal and postpubertal development in gilts

The crossbred gilts studied were aged 80 days (prepubertal), 180 days (prepubertal or postpubertal) and 260 days (postpubertal or pregnant). Estimates of metabolic clearance rate (MCR) of oestradiol and progesterone were consistently less (21 and 27%) in plasma than in blood, and these differences were not influenced by age of gilt. The MCR (1/day per kg body weight) for oestradiol and progesterone in plasma was greater (P less than 0.05) for 80-day-old prepubertal gilts than for older gilts. The MCR values of oestradiol and progesterone were similar in 180-day-old and 260-day-old gilts independent of reproductive state. Production rate (PR) of oestradiol and progesterone increased with age (80-180 days), and age and reproductive state differences were much more pronounced for PR of progesterone than of oestradiol. These results support the hypothesis that a reduction in the MCR and an increase in PR of oestradiol and progesterone in the gilt are associated with the process of pubertal development, and changes in gonadal steroid concentrations appear not to alter the MCR of oestradiol and progesterone.