Land-use history of the Axlarp area in the Småland uplands,southern Sweden: palaeoecological and archaeological investigations

The results of pollen analysis, magnetic measurements (SIRM), and archaeological and historical investigations, in the Axlarp area are presented. With respect to natural conditions and the distribution of prehistoric features, this area is typical of the higher parts of the Småland uplands, which, agriculturally, is a marginal region of southern Sweden. The study shows that farming in the Axlarp area began at ca. 700 B.C. (dates in calibrated/calendar years). The period 700 B.C.-A.D. 500 was characterized by shifting cultivation of Hordeum and Triticum and much pasture. Between A.D. 500 and A.D. 1200 farming declined but some pasturage was still practised, possibly on a seasonal basis. Two farms were established in the Middle Ages, probably between A.D. 1200–1300. Cereals were sown in stone-cleared fields and pastoral farming and hay making was carried out. One farm was deserted during the 15th or early 16th century and the other developed into the hamlet Axlarp whose farmers practised a three-course cropping system. Land-use history as recorded in the pollen diagram can be related to activities associated with these farms. Cereals grown after A.D. 1200 included Hordeum and Avena, and possibly Triticum and Secale. There are no indications of slash-and-burn cultivation in the area.     

A Lipoprotein Receptor Cluster IV Mutant Preferentially Binds Amyloid-β and Regulates Its Clearance from the Mouse Brain

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ∼70% of amyloid β-peptide (Aβ) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma. Experimental studies have shown that free circulating Aβ re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aβ from entering the brain. Treatment of Alzheimer APPsw^+/0 mice with WT-LRPIV has been shown to reduce brain Aβ pathology. In addition to Aβ, LRPIV binds multiple ligands. To enhance LRPIV binding for Aβ relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aβ40 and Aβ42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3–1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aβ40 and Aβ42 25–27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw^+/0 mice with LRPIV-D3674G (40 μg/kg/day) reduced Aβ40 and Αβ42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60–80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aβ clearance therapy.     

The pivotal role of hepatocytes in drug discovery

This review promotes the value of isolated hepatocytes in modern Drug Discovery programmes and outlines how increased understanding, particularly in the area of in vitro-in vivo extrapolation (IVIVE), has led to more widespread use. The importance of in vitro metabolic intrinsic clearance data for predicting in vivo clearance has been acknowledged for several years and the greater utility of hepatocytes, compared with hepatic microsomes and liver slices, for this application is discussed. The application of hepatocytes in predicting drug-drug interactions (DDIs) resulting from reversible and irreversible (time-dependent) inhibition is relatively novel but affords the potential to study both phase I and phase II processes together with any impact of drug efflux and/or uptake (cellular accumulation). Progress in this area is reviewed along with current opinions on the comparative use of primary hepatocytes and higher throughput reporter gene-based systems for studying cytochrome P450 (CYP) induction. The appreciation of the role of transporter proteins in drug disposition continues to evolve. The study of hepatic uptake using isolated hepatocytes and the interplay between drug transport and metabolism with respect to both clearance and DDIs and subsequent IVIVE is also considered.     

The diurnal rhythms of cholesterol metabolism and plasma clearance of model chylomicrons: comparison of normal and genetically hypercholesterolemic rats (RICO)

Previous studies showed a slower clearance of cholesterol-labeled lymph chylomicrons in genetically hypercholesterolemic rats (RICO) compared with normocholesterolemic rats. In this study, we compared rates of lipolysis and remnant clearance in RICO versus control normocholesterolemic rats of the same strain (RAIF) or with control Wistar rats, by injecting chylomicron-like lipid emulsions labeled with ¹⁴C-triolein to trace lipolysis, and ³H-cholesteryl ester to trace remnant clearance. Our findings showed slower clearance of chylomicron remnants in RICO compared with control RAIF or with control Wistar rats. During the light period, the clearance of lipids from chylomicron-like lipid emulsions injected intravenously was significantly slower in RICO rats compared with normocholesterolemic control rats of the same strain, RAIF. Within the RICO group, clearance of emulsion triolein (TO) was faster during the dark period compared with the light period. In contrast, however, the clearance of the emulsion remnants traced by cholesteryl oleate (CO) was slower during the dark period. This behaviour was not found within the Wistar group, where the clearances of TO and CO were similar in the light and dark period. Hepatic clearance of chylomicron remnants is mediated primarily by the low density lipoprotein (LDL) receptor, the expression of which shows diurnal variation. In both Wistar and RICO rats, the expression of LDL receptors was highest during the dark period. The LDL receptors in hepatic microsomal membranes from RICO rats migrated faster on SDS polyacrylamide gel electrophoresis when compared with normal Wistar and the RAIF. However in hepatic plasma membranes the LDL receptors from RICO and Wistar rats appeared identical after immunoblotting. Furthermore the LDL receptors from RICO and Wistar rats responded similarly to treatment with neuraminidase. An alteration in post-translational processing of the LDL receptor could possibly account for the slower clearance of chylomicron remnants in the RICO.     

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

Fate of fluorescent microspheres in developing Ictalurus punctatus following prolonged immersion

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes (p < 0.05). Significantly higher FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs (p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.     

Effects of seston variability on the clearance rate and absorption efficiency of the mussels Aulacomya maoriana, Mytilus galloprovincialis and Perna canaliculus from New Zealand

The clearance rates (CR in l g⁻¹ h⁻¹) and net absorption efficiencies (AE) of three co-occurring mytilid species were estimated to determine the effects of variation in ambient seston quantity (total particulate matter, TPM, mg l⁻¹) and quality (particulate organic matter, POM, mg l⁻¹; percent organic matter, PCOM=POM/TPM) on these physiological functions. CR_S estimates were significantly different among the species (Mytilus galloprovincialis>Perna canaliculus=Aulacomya maoriana), but were not correlated with differences in species-dependent mean body size (P. canaliculus>M. galloprovincialis>A. maoriana). AE estimates were independent of mean body weight, and did not differ among the species. For all three species, CR responded in a simple linear manner to seston organic content, either in terms of POM (for A. maoriana and M. galloprovincialis), or PCOM (for P. canaliculus). The AE response, although also of a simple linear type, was species-dependent and involved interactions of two or three seston components: TPM, POM and PCOM for A. maoriana; POM and PCOM for M. galloprovincialis; and TPM and PCOM for P. canaliculus. For all three species, seston variation explained 15-20% of the variation in CR and 52-59% of the variation in AE. Comparisons among the species indicated that two very different responses to variation in seston quantity and quality exist. First, significant differences in CR result from species-dependent differences in the magnitude of the response to seston variation. Second, species-dependent responses to variation in seston variation resulted in the similarity of the AE responses. Thus, the three species appear to have evolved different strategies for dealing with seston variation, but with the end result that their AE responses do not differ. Finally, no evidence was found of a negative association between CR and AE for any of the three species, suggesting that under the seston conditions experienced in this work, these species do not have the physiological compensatory capacity to reduce CR in order to increase AE. The importance of a comparative (i.e., multi-species) approach utilising ambient seston is emphasised by the findings of this research if we are to understand better the feeding and digestive physiologies of suspension-feeding organisms such as mussels.     

Secondary necrosis in multicellular animals: an outcome of apoptosis with pathogenic implications

In metazoans apoptosis is a major physiological process of cell elimination during development and in tissue homeostasis and can be involved in pathological situations. In vitro, apoptosis proceeds through an execution phase during which cell dismantling is initiated, with or without fragmentation into apoptotic bodies, but with maintenance of a near-to-intact cytoplasmic membrane, followed by a transition to a necrotic cell elimination traditionally called “secondary necrosis”. Secondary necrosis involves activation of self-hydrolytic enzymes, and swelling of the cell or of the apoptotic bodies, generalized and irreparable damage to the cytoplasmic membrane, and culminates with cell disruption. In vivo, under normal conditions, the elimination of apoptosing cells or apoptotic bodies is by removal through engulfment by scavengers prompted by the exposure of engulfment signals during the execution phase of apoptosis; if this removal fails progression to secondary necrosis ensues as in the in vitro situation. In vivo secondary necrosis occurs when massive apoptosis overwhelms the available scavenging capacity, or when the scavenger mechanism is directly impaired, and may result in leakage of the cell contents with induction of tissue injury and inflammatory and autoimmune responses. Several disorders where secondary necrosis has been implicated as a pathogenic mechanism will be reviewed.     

Impact of the zebra mussel Dreissena polymorpha invasion on the budget of suspended material in a shallow lagoon ecosystem

The role of the zebra mussel Dreissena polymorpha in redistribution of total particulate material (TPM) between the water column and bottom sediment was estimated using the TPM budget for a mussel bed in the Curonian lagoon, the Baltic Sea. Seasonal clearance rates were derived from the TPM budget assuming two resuspension scenarios: no resuspension and full resuspension of biodeposits. Estimated clearance rates for both scenarios were compared with the rates calculated from the population clearance rate model. Seasonal clearance rates estimated using the population model (1.1 and 11.8 l g⁻¹ SFDW day⁻¹) fitted well into the interval of seasonal clearance rates calculated from TPM budgets assuming no resuspension of biodeposits (3.2 and 21.4 l g SFDW⁻¹ day⁻¹). In the scenario with biodeposits resuspension clearance rates were much higher (57.4 and 148.9 g SFDW⁻¹ day⁻¹). The ratio of clearance to residence time was highly dependent on the fate of biodeposits. Therefore its use in interpretation of the species impact on TPM was limited. An alternative measure based on the ratio of the amount of TPM biodeposited to TPM transported into the bed was used. It was found that zebra mussels are able to deposit between 10 and 30% of the incoming TPM, and the amount of biodeposited material was correlated with water residence time. Results indicate that the impact of zebra mussels on TPM in the lagoon is small relative to the high transport rates of TPM over the bed. However, annual biosedimentation rate (~590 g m⁻²) in the mussel bed was higher than physical deposition rate (~380 g m⁻²) in accumulation areas devoid of large suspension feeders. We suggest that a local impact due to enhanced availability of organic material to other trophic groups of associated benthic organisms may be more significant than effects on TPM pathways at an ecosystem scale.     

Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.