Physico-chemical properties of the N-terminally truncated L68Q cystatin C found in amyloid deposits of brain haemorrhage patients

Cystatin C, a major extracellular cysteine proteinase inhibitor, is deposited as amyloid in brain haemorrhage patients with hereditary cystatin C amyloid angiopathy (HCCAA). A disease-causing mutation on the genetic level results in the substitution Leu68-->Gln (L68Q) in cystatin C, which causes protein instability. Besides carrying the L68Q substitution, cystatin C in amyloid deposits isolated from patients is N-terminally truncated by 10 amino acids. To elucidate the role of the N-terminal truncation for protein stability and aggregation properties, (delta1-10,L68Q)-cystatin C was produced in an Escherichia coli expression system and characterised. Unlike wild-type cystatin C, this variant rapidly dimerised under physiological conditions. Two unfolding intermediates of (delta1-10,L68Q)-cystatin C were identified, under the same pH and ionic strength conditions as required to form intermediates of full-length L68Q cystatin C. No evidence was found that the N-terminal truncation per se alters protein stability and leads to higher forms of aggregation. Monomeric as well as dimeric L68Q cystatin C incubated with neutrophil elastase was truncated as in HCCAA patients' amyloid. A protein variant with a thrombin cleavage site placed in front of residue Gly11 in L68Q cystatin C was constructed and used to confirm that the N-terminal segment is similarly accessible to proteinases in the monomeric and dimeric states of L68Q cystatin C. Thus, the N-terminal segment of L68Q cystatin C is exposed to proteolytic attack and does not seem to be involved in intramolecular contacts leading to dimerisation or higher-order aggregation. We conclude that the N-terminal truncation likely is an event secondary to amyloid formation, and of no relevance for the development of HCCAA.     

Gall-inducing insects provide insights into plant systematic relationships

Field surveys of cynipid gall-inducer occurrences on Quercus species were conducted in Florida, North Carolina, and Pennsylvania, USA. All cynipids demonstrated strong host species and organ fidelity. One result of this specialization is effective niche partitioning among cynipids. The host-association patterns of these specialist herbivores should reflect similarities among oaks, thus we clustered oak species according to their cynipid distributions. Cynipids distinguished small differences among their hosts. A dendrogram of oak species based on cynipid distributions was largely congruent with botanical arrangernents. Cynipid occurrences offer information helpful to resolving some aspects of oak systematics. Collaborative efforts between taxonomic botanists and entomologists will be useful in resolving a variety of plant and insect systematic problems.     

PATTERNS OF RESOURCE ALLOCATION IN WILDFLOWER POPULATIONS OF FIELDS AND WOODS

Fifty-two populations of fifty species of wildflowers characteristic of either field or deciduous forest were analyzed to determine pattens of biomass allocation to component organs. These populations' allocation patterns were used to determine generalized allocation patterns of the herb component of earlier (field) and later (deciduous forest) secondary successional communities. The following patterns were determined: (1) The proportion of dry matter allocated to seed reproductive organs was greater in field populations than in woods populations; (2) The herbs of woodland habitats allocated a greater proportion of their resources to leaves and belowground organs than field habitat herbs; (3) The field annuals allocated a greater proportion of their resources to reproduction than field perennials; (4) Introduced species showed a higher reproductive allocation than native species of fields; (5) Regression analysis showed strong correlations of component organ biomass to total biomass and belowground biomass to shoot biomass.     

Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.     

Serglycin expression during monocytic differentiation of U937-1 cells

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.      

BEHAVIORAL EVIDENCE FOR HOST-RACE FORMATION IN EUROSTA SOLIDAGINIS

We report behavioral evidence that Eurosta solidaginis, a stem-galling tephritid fly, has formed host races on its two goldenrod hosts, Solidago altissima and S. gigantea. Previous work has shown that flies from each host plant differ electrophoretically at the level of host races. The two host-associated populations were truly sympatric and were frequently found on host plants of the two species growing interdigitated with each other. Each host-associated population demonstrated a strong preference for ovipuncturing its own host. The S. gigantea–associated population emerged 10 to 14 d earlier than the S. altissima–associated population, contributing to the reproductive isolation between populations. Partial reproductive isolation is also maintained by a preference for mating on the host from which the fly emerged. The populations meet the criteria established for host races, suggesting that they may be in an intermediate stage of sympatric speciation.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.     

The pattern,rate, and range of within‐patch movement of a stem‐galling fly

1. Information on the movement of insects is critical to understanding the spatial spread, dynamics, and genetic structure of their populations, as well as their interactions with other species. With this in mind, the movement behaviour of the stem‐galling fly Eurosta solidaginis Fitch (Diptera: Tephritidae) was investigated. 2. Fluorescent‐marked adults were released at a single location within pure patches of the host plant, tall goldenrod Solidago altissima, and their distributions censused repeatedly throughout the day. 3. Following their release, male and female flies redistributed themselves in a manner that was well described by a simple‐diffusion model. The diffusion rate was independent of fly density and time since flies were released. 4. Female flies dispersed at a significantly faster rate, and therefore farther on average, than males. Based on the diffusion model, it was estimated that at 2.5–3.0 h post release, males and females had a median dispersal distance of only 2.0 and 2.5 m respectively. Furthermore, 95% of the males were estimated to have dispersed no more than 5.9 m, and females no more than 6.4 m. 5. Post‐release censuses suggested that flies were most active during mid morning, disappeared from the site at a rate of 10–15% per hour (most likely due to mortality), and survived for less than 2 days. Based on the rate of spread, diel activity, and liberal estimates of longevity in the field, 50% of the ovipositing females were predicted to have had a maximum lifetime range of movement within a patch of hosts of ≤ 51 m (95% were expected to have been limited to ≤ 130 m). 6. These data are used to assess whether the absence of a positive correlation between host‐plant preference and offspring performance in this system could be due to the limited scale of dispersal of this species relative to the spatial scale at which its oviposition behaviour has been studied.