Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor’s action.     

Carnivore involvement in bone assemblages based on taphonomic and zooarchaeological analyses of Cova del Coll Verdaguer site (Barcelona,Iberian Peninsula)

Abstract

Carnivores are the main biological agents identified in the Upper Pleistocene assemblage of Cova del Coll Verdaguer (NE Iberian Peninsula). At the site, ungulate bones were primarily accumulated and modified by hyenas, as shown by partly digested bones, coprolites and the pattern of damage on bones. However, other small- to medium-sized carnivores seemed to be involved in the accumulation, owing primarily to the presence of their bones and coprolites. Finally, other carnivores, including the brown bear, used the cave to hibernate and for birthing cubs. The combination of zooarchaeological and taphonomic studies, together with previous coprogenic analyses, at the site indicates that it is only by using these combined approaches that it is possible to identify the majority of the agents involved in an accumulation.     

Ontogenesis of alpha-amylase in rat parotid gland during postnatal development

Summary Changes in -amylase (-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of -amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage.Career scientist of the Consejo Nacional de Investigaciones Científicas y Téchnicas of Argentina     

Biased mutations and microsatellite variation

Mutation bias is one of the forces that may constrain the variation at microsatellite loci. Here, we study the dynamics of population statistics and the genetic distance between two populations under multiple stepwise mutations with linear bias and random drift. Expressions are derived for these statistics as functions of time, as well as at mutation-drift equilibrium. Applying these expressions to published data on humans and chimpanzees, the regression coefficient of mutation bias on allele size was estimated to be at least between - 0.0064 and -0.013. The assumption of mutational bias produces larger estimates of divergence times than are obtained in its absence; in particular, the time of split between African and non-African human populations is estimated to be between 183,000 and 222,000 years, assuming one-step mutations and no selection. With multistep mutations, the divergence time is estimated to be lower.      

Experimentally induced clutch size enlargements affect reproductive success in the Pied Flycatcher

We performed a food provisioning experiment in a population of Pied Flycatchers Ficedula hypoleuca breeding at high altitude in central Spain to test if food availability before and during laying determines clutch size. Food was provided to one of two pairs with the same date of initiation of nest-building (15 dyads of subsequently reproducing pairs were thus created). Food provisioning began on the day of initiation of nest-building and ended on the day after the last egg was laid. Although laying date was unaffected by the experiment, clutch size in the experimental treatment was significantly larger. This result could indicate that food availability at laying (1) proximately constrained clutch size or (2) that females evaluated future conditions for incubating eggs and feeding nestlings based on food availability at laying. Reproductive success (proportion of eggs that resulted in fledged young) was significantly reduced in the experimental treatment. This effect suggest that supplemented females were tricked by the experiment into laying more eggs than the number of eggs they were able to incubate with success and the number of nestlings they were able to feed, a source of error in clutch size adjustment which could be common in non-experimental situations.     

Heterogeneous responses to ozone and nitrogen alter the species composition of Mediterranean annual pastures

Air pollution represents a threat to biodiversity throughout the world and particularly in the Mediterranean area, where high tropospheric ozone (O₃) concentrations and atmospheric nitrogen (N) deposition are frequently recorded. Mediterranean annual pastures are among the most important ecosystems in southern Europe due to their high biodiversity and extension. Aiming to study the responses of these communities to the main atmospheric pollutants in the Mediterranean region, an experimental study was performed in an open-top chamber (OTC) facility. A mixture of six species representative of annual pastures was grown under field conditions inside the OTC. Plants were exposed for 39 days to four O₃ treatments and three doses of N. The species responded heterogeneously to both factors. Legumes did not react to N but were very sensitive to O₃: Trifolium species responded negatively, while Ornithopus responded positively, taking advantage of the greater sensitivity of clovers to O₃. The grasses and the herb were more tolerant of O₃ and grasses were the most responsive to N. Significant interactions between factors indicated a loss of effectiveness of N in O₃-polluted atmospheres and an ability of O₃ to counterbalance the damage induced by N input, but both effects were dependent on O₃ and N levels. The inclusion of plant competition in the experimental design was necessary to reveal results that would otherwise be missed, such as the positive growth responses under elevated O₃ levels. Surprisingly, competition within the legume family played the most important role in the overall response of the annual community to O₃. Both tropospheric O₃ and N deposition should be considered important drivers of the structure and biodiversity of Mediterranean annual pastures.     

Role of nucleotide‐binding oligomerization domain 1 (NOD1) in pericyte‐mediated vascular inflammation

We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll‐like receptor 4 (TLR4). However, Gram‐negative pathogen‐associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co‐purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12‐iE‐DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12‐iE‐DAP‐dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF‐κB appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram‐negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management.     

The invasion of <Emphasis Type="Italic">Senecio pterophorus</Emphasis> across continents: multiple,independent introductions,admixture and hybridization

Senecio pterophorus (Compositae) is a perennial shrub native to eastern South Africa that was introduced into the Western Cape in South Africa and Australia approximately 100 years ago and into Europe (Italy and Spain) more than 25–30 years ago. In this study, the aims were to unravel the putative sources of the introduced populations and identify the changes in genetic diversity after invasion using molecular markers and phylogeographic and population genetic analyses. We sampled the entire area of distribution for S. pterophorus extensively. Based on the results, three lineages were established along a latitudinal and climatic gradient in the native range (south, central, central/north) with high levels of admixture. Multiple, independent introductions occurred in the four invaded ranges. The central/northern lineage (humid climate) was the primary source for all of the invaded regions (with drier climates), although a secondary role was revealed for the southern lineage in the Western Cape and the central/northern lineage in Australia and Spain. The genetic diversity was slightly lower in the Spanish and Australian populations than that in the native populations. A variety of demographic and genetic processes affected the amount and structure of genetic diversity in the invaded areas, including multiple introductions and admixture (Western Cape, Australia and Spain) as well as pre-invasive hybridization (Italy). The patterns of dispersion support a hypothesis of rapid evolution of S. pterophorus after invasion in response to novel climatic conditions.     

Kinetics and structural requirements for the binding protein of the Di-tripeptide transport system of Lactococcus lactis

The gene (dppA) encoding the binding protein of the di-tripeptide ABC transporter of Lactococcus lactis (DppA) was cloned under the control of the nisin promoter. Amplified expression ( approximately 200-fold increase) of the protein fused to a carboxyl-terminal six-histidine tag allowed the purification of DppA-(His)(6) by nickel-chelate affinity and anion-exchange chromatography. Ligand binding to DppA-(His)(6) elicited an electrophoretic mobility shift, a decrease in the intrinsic fluorescence, and a blue shift of the emission maximum. Each of these parameters detected conformational changes in the protein that reflect ligand binding, and these were used to determine the structural requirements of DppA-(His)(6) for binding peptides. The major features of peptide binding include (i) high affinity for di- and tripeptides, (ii) requirement of a free N-terminal alpha-amino group and an alpha-peptide bound contiguous with the N-terminal amino group, (iii) stereospecificity for L-isomers, and (iv) preference for dipeptides containing methionine or arginine, followed by hydrophobic tripeptides consisting of leucine or valine residues. Maximal binding affinity was detected at pH 6.0, and the K(d) for binding increased 1 order of magnitude for every unit increase in pH. This suggests that the ionization of protein residues (pK > 6.0) in or in close proximity to the binding site is critical in the binding mechanism.