A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation

A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli. The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature-inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.     

Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system

A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Forward Modeling of Fluctuating Dietary 13C Signals to Validate 13C Turnover Models of Milk and Milk Components from a Diet-Switch Experiment

Isotopic variation of food stuffs propagates through trophic systems. But, this variation is dampened in each trophic step, due to buffering effects of metabolic and storage pools. Thus, understanding of isotopic variation in trophic systems requires knowledge of isotopic turnover. In animals, turnover is usually quantified in diet-switch experiments in controlled conditions. Such experiments usually involve changes in diet chemical composition, which may affect turnover. Furthermore, it is uncertain if diet-switch based turnover models are applicable under conditions with randomly fluctuating dietary input signals. Here, we investigate if turnover information derived from diet-switch experiments with dairy cows can predict the isotopic composition of metabolic products (milk, milk components and feces) under natural fluctuations of dietary isotope and chemical composition. First, a diet-switch from a C₃-grass/maize diet to a pure C₃-grass diet was used to quantify carbon turnover in whole milk, lactose, casein, milk fat and feces. Data were analyzed with a compartmental mixed effects model, which allowed for multiple pools and intra-population variability, and included a delay between feed ingestion and first tracer appearance in outputs. The delay for milk components and whole milk was ∼12 h, and that of feces ∼20 h. The half-life (t_½) for carbon in the feces was 9 h, while lactose, casein and milk fat had a t_½ of 10, 18 and 19 h. The ¹³C kinetics of whole milk revealed two pools, a fast pool with a t_½ of 10 h (likely representing lactose), and a slower pool with a t_½ of 21 h (likely including casein and milk fat). The diet-switch based turnover information provided a precise prediction (RMSE ∼0.2 ‰) of the natural ¹³C fluctuations in outputs during a 30 days-long period when cows ingested a pure C3 grass with naturally fluctuating isotope composition.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Structural, functional, and evolutionary characterization of novel members of the allatostatin receptor family from insects

By using degenerate primers based on known mammalian somatostatin receptors and the recently identified Drosophila allatostatin receptors (AlstR), we have cloned a novel receptor for the neuropeptide, allatostatin, from the cockroach Periplaneta americana. The receptor exhibits about 60% amino acid identity in the transmembrane regions when compared to the two known AlstRs from Drosophila melanogaster. In addition, two cDNA fragments were obtained from the stick insect Carausius morosus, one of which is similar to Drosophila AlstR, whereas the other is more similar to mammalian somatostatin receptors. Functional expression in Xenopus oocytes shows that the Periplaneta-AlstR exhibits high affinity to endogenous cockroach allatostatin peptides. Studies with synthetic peptides demonstrate that agonistic activity is mediated by the conserved C-terminal pentapeptide YXFGL-amide; in this sequence, amidation of the C-terminus is obligatory to maintain affinity. Thus, our studies provide a molecular basis for understanding the widespread biological activities of the allatostatin peptides.     

Effects of metabolic neuropeptides from insect corpora cardiaca on proline metabolism of the African fruit beetle,Pachnoda sinuata

The effect of neuropeptides from the corpora cardiaca of the fruit beetle Pachnoda sinuata on proline metabolism has been investigated in vivo. Conspecific injections of a crude extract from corpora cardiaca cause an increase of the concentration of proline in the haemolymph by nearly 20% and a decrease of the concentration of alanine, the precursor in proline synthesis, by about 64% when compared with a water-injected group. Purification of an extract of corpora cardiaca on reversed-phase liquid chromatography revealed two distinct UV absorbance and fluorescence peaks that cause hyperprolinaemia in the fruit beetle. The major peak is the previously identified octapeptide Mem-CC; the second peak is also a peptide, but its primary sequence remains, as yet, unidentified. Synthetic Mem-CC elicited time- and dose-dependent increases/decreases of the concentrations of proline and alanine in the haemolymph respectively. Furthermore, the receptor for this peptide seems to be specific in P. sinuata: only peptides of the large family of adipokinetic hormones with an Asp, Asn or Gly residue at position 7 could elicit biological activity, whereas those with a Trp, Ser or Val residue at this position did not have any activity.     

Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.