Ig heavy chain of the sturgeon Acipenser baeri : cDNA sequence and diversity

 To investigate the gene organization of the IGH locus, and the VH diversity of the Siberian sturgeon, a cDNA library was constructed and screened with VH-specific probes from two holostean fish. Isolated clones were analyzed and domain-specific probes used in rescreening of the library, Southern blot analysis, and northern blots. It was concluded that the Siberian sturgeon has one IGH locus with a translocon type of organization. Two allelic variants of the mu gene were found, with identities ranging from 80 to 100% for the different domains (highest for CH4 and lowest for CH2). Sturgeon CH sequences are most closely related to those of holostean fish. There are three distinct VH families, VHI grouping with mammalian clan III, VHII grouping with the teleost clan, and VHIII grouping with the archaic clan. The variability of the CDR 3 region is substantial, and we identified a number of conserved motifs in the D segment. Further, we deduced that there are at least nine different JH segments in the locus, contributing to the antibody repertoire of the sturgeon. The variable segments of the three families can be associated with any of the D or JH segments in the rearrangement. Sturgeon, in addition to the random rearrangement of VH, D, and JH segments, have exonuclease activity, and an introduction of N and probably P nucleotides at the site of rearrangement. Received: 2 March 1998 / Revised: 20 May 1998     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Effect of Serine and Ethanolamine Administration on Phospholipid-Related Compounds and Neurotransmitter Amino Acids in the Rabbit Hippocampus

Abstract: The report concerns mechanisms for the increase of extracellular levels of ethanolamine and phosphoethanolamine in CNS regions, such as the hippocampus, in transient brain ischemia, hypoglycemia, seizures, etc. l -Serine (2.5–10 m M ), d -serine (10 m M ), or ethanolamine (10 m M ) was administered for 20 min via a microdialysis tubing to the hippocampus of unanesthetized rabbits. The concentrations of primary amines were determined in the dialysates. When levels were elevated 10–100 times in the extracellular fluid, l -serine caused a dose-dependent increase of the concentration of extracellular ethanolamine. Ethanolamine caused a corresponding, although somewhat smaller, increase in serine levels. Furthermore, l -serine also induced an increased concentration of phosphoethanolamine that was delayed in time relative to the peak of ethanolamine. d -Serine was as effective as l -serine in raising ethanolamine levels but had no effect on phosphoethanolamine. Ethanolamine, but not l -serine, also increased extracellular glutamate/aspartate levels in an MK-801-dependent fashion. A similar effect, but delayed in time, was observed with d -serine. These effects were inhibited by MK-801. The concentrations of other amino acids were not significantly affected. The characteristics of the effects are suggestive of base exchange reactions between serine and ethanolamine and between ethanolamine and serine glycerophospholipids, respectively, in neuronal plasma membranes.     

Error Rates and Polymorphism Frequencies for Three RAPD Protocols

Three amplification protocols were analyzed for error rate and generation of polymorphisms during RAPD analysis. Using a set of 240 primers, the protocols detected similar frequencies of polymorphisms in two inbred sugar beet lines. The error rate was investigated by including a 1:1 mixture of DNA from the two lines in all analyses. Similar error rates, approximately 18%, were detected by the three protocols. Thus, altered amplification conditions did not substantially affect the error rate during RAPD analysis. For each of the three possible pairs of protocols, a positive correlation was obtained for primer and number of polymorphisms. Thus, a set of highly polymorphic RAPD primers can be used effectively, without prior screening, to detect polymorphisms for each protocol.     

Arrest of chlorophyll accumulation prior to anthocyanin formation in Euphorbia pulcherrima

Euphorbia pulcherrima Klotz plants exposed to short days (11 h light/13 h dark) for a period of eight weeks, developed flowers and a red canopy consisting of bracts and few true leaves. Plants maintained for three weeks under short day conditions, failed to produce flower primordia or a red canopy in the following 5 weeks in continuous light. Between the 4th and 5th short day week, the youngest leaves began to accumulate anthocyanin and turned red while the apical meristems differentiated into flower primordia. Chlorophyll accumulation ceased at the onset of anthocyanin synthesis and the protein content per unit leaf area declined. mRNA for glutamyl-tRNA^Glu synthetase (EC 6.1.1.17) and glutamyl-tRNA^Glu reductase also declined during this period. Western blot analysis revealed a loss of glutamyl-tRNA^Glu reductase, glutamate 1-semialdehyde (EC 5.4.3.8) and the Mg-chelatase subunits, Olive and CH42, in the last 2 to 4 weeks of the photoperiod.     

Changes in organic phosphorus composition in boreal forest humus soils: the role of iron and aluminium

Organic phosphorus (P) is an important component of boreal forest humus soils, and its concentration has been found to be closely related to the concentration of iron (Fe) and aluminium (Al). We used solution and solid state ³¹P NMR spectroscopy on humus soils to characterize organic P along two groundwater recharge and discharge gradients in Fennoscandian boreal forest, which are also P sorption gradients due to differences in aluminium (Al) and iron (Fe) concentration in the humus. The composition of organic P changed sharply along the gradients. Phosphate diesters and their degradation products, as well as polyphosphates, were proportionally more abundant in low Al and Fe sites, whereas phosphate monoesters such as myo-, scyllo- and unknown inositol phosphates dominated in high Al and Fe soils. The concentration of inositol phosphates, but not that of diesters, was positively related to Al and Fe concentration in the humus soil. Overall, in high Al and Fe sites the composition of organic P seemed to be closely associated with stabilization processes, whereas in low Al and Fe sites it more closely reflected inputs of organic P, given the dominance of diesters which are generally assumed to constitute the bulk of organic P inputs to the soil. These gradients encompass the broad variation in soil properties detected in the wider Fennoscandian boreal forest landscape, as such our findings provide insight into the factors controlling P biogeochemistry in the region but should be of relevance to boreal forests elsewhere.     

Binding of netrin-4 to laminin short arms regulates basement membrane assembly

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.     

Histamine-like immunoreactivity in photoreceptors of the compound eyes and ocelli of the flies Calliphora erythrocephala and Musca domestica

Summary Antibodies to histamine were used for immunocytochemical studies of the visual system in the flies Calliphora erythrocephala and Musca domestica. Specific immunolabeling of photoreceptors was found both in the compound eyes and ocelli of both species. In the compound eyes histamine-like immunoreactivity (HA-IR) was found in all the short visual fibers (photoreceptors R1–6) and one type of long visual fiber (photoreceptor R8). In addition, the ocellar photoreceptors also show HA-IR. In view of earlier biochemical and pharmacological/physiological findings by Elias and Evans (1983) and Hardie (1987) it thus seems likely that histamine is a neurotransmitter in insect photoreceptors. Interestingly, the second type of long visual fiber (photoreceptor R7) has recently been found to be GABA-immunoreactive (Datum et al. 1986). The two types of long visual fibers may hence use different transmitters which act on different receptors of the postsynaptic neurons in the second visual neuropil, the medulla. In addition to the photoreceptors in the retina and ocelli, we found processes of HA-IR neurons in one of the optic lobe neuropils, the lobula. This finding indicates that histamine may also be a transmitter in certain interneurons in the visual system.Abbreviations HA histamine - GABA -amino butyric acid - GAD glutamic acid decarboxylase - 5-HT 5-hydroxytryptamine (serotonin) - HA-IR histamine-like immunoreactivity - R1-R6 class of short-axoned photoreceptors - R7 and R8 long-axoned photoreceptors - LMC large monopolar neuron of lamina - HSA human serum albumin - PBS phosphate-buffered saline - DEPC diethylpyrocarbonate     

Gross primary production controls the subsequent winter CO2 exchange in a boreal peatland

In high‐latitude regions, carbon dioxide (CO₂) emissions during the winter represent an important component of the annual ecosystem carbon budget; however, the mechanisms that control the winter CO₂ emissions are currently not well understood. It has been suggested that substrate availability from soil labile carbon pools is a main driver of winter CO₂ emissions. In ecosystems that are dominated by annual herbaceous plants, much of the biomass produced during the summer is likely to contribute to the soil labile carbon pool through litter fall and root senescence in the autumn. Thus, the summer carbon uptake in the ecosystem may have a significant influence on the subsequent winter CO₂ emissions. To test this hypothesis, we conducted a plot‐scale shading experiment in a boreal peatland to reduce the gross primary production (GPP) during the growing season. At the growing season peak, vascular plant biomass in the shaded plots was half that in the control plots. During the subsequent winter, the mean CO₂ emission rates were 21% lower in the shaded plots than in the control plots. In addition, long‐term (2001–2012) eddy covariance data from the same site showed a strong correlation between the GPP (particularly the late summer and autumn GPP) and the subsequent winter net ecosystem CO₂ exchange (NEE). In contrast, abiotic factors during the winter could not explain the interannual variation in the cumulative winter NEE. Our study demonstrates the presence of a cross‐seasonal link between the growing season biotic processes and winter CO₂ emissions, which has important implications for predicting winter CO₂ emission dynamics in response to future climate change.     

Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state

The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of matrilins and potentially of other extracellular matrix proteins.