Chelonus sp. near curvimaculatus venom proteins: Analysis of their potential role and processing during development of host Trichoplusia ni

The venom that Chelonus sp. near curvimaculatus injects into each parasitized Trichoplusia ni egg is entirely injected within the first 8 s of the 19-s oviposition period, before deposition of the parasitoid egg that is injected during the final 1-2 s of the oviposition. The parasitization factor, causing precocious metamorphosis of the host, is injected after the venom, but before the parasite egg. The venom by itself does not cause developmental redirection of the host. Chelonus venom proteins are very stable in the host egg during the first 2 days of egg development. Then, on the last day before hatching, they are rapidly degraded by the proteolytic enzymes appearing in 3-day-old T. ni eggs. Among those that degrade the venom proteins are serine-type proteinases, and at least one seems to be a trypsin-like enzyme.     

Endocrine interrelationship between the parasitoid Chelonus sp. and its host Trichoplusia ni

The egg-larval parasitoid Chelonus sp. induces the precocious onset of metamorphosis in the 4th (penultimate) stadium of its host Trichoplusia ni, emerges from the prepupa, and then feeds on it. Qualitative and quantitative changes in ecdysteroids and juvenile hormone were measured. Hemolymph of 3rd-to 4th-instar host larvae and the parasitoids they contained, as well as nonparasitized and parasitized eggs, were analyzed. In the host hemolymph a broad peak of ecdysteroids during molting into the 4th stadium and a continuous increase from day 2 (onset of precocious wandering) until day 4 (emergence of parasitoid) were observed; 20-hydroxyecdysone and 20,26-dihydroxyecdysone were predominant. The juvenile hormone titer fluctuated in the 3rd and early 4th stadium and fell to undetectable levels shortly before the precocious onset of wandering. The parasitoid's ecdysteroids started to increase on the molt to the 2nd instar (= early 4th instar of the host) and thereafter fluctuated on a high level, 20-hydroxyecdysone, 20,26-dihydroxy-ecdysone, and ecdysone being predominant. The juvenile hormone titer was high in late 1st-instar parasitoids, decreased to low levels at ecdysis into the 2nd instar, and increased again to high levels in the 2nd-instar larvae at the time when their shape changed from flat to cylindrical. After ecdysis to the 3rd instar the juvenile hormone titer fell. A comparison revealed that both ecdysteroids and juvenile hormone fluctuate independently in parasitoid and host at most stages, suggesting that the parasitoid produces its own hormones. The first data on ecdysteroids and juvenile hormones in the egg stage of a parasitoid/host system are reported. At the stage of eye pigmentation parasitized eggs contained more immunoreactive midpolar ecdysteroids than non-parasitized ones. 20-Hydroxyecdysone and 20,26-dihydroxyecdysone were the predominant ecdysteroids in both nonparasitized and parasitized eggs, but the latter contained several additional ecdysteroids which were not seen in nonparasitized eggs. The titer of juvenile hormone was similar in both. Shortly before hatching the ecdysteroids were low in parasitized and nonparasitized eggs, but the content of juvenile hormone was much higher in the former. At this stage the majority of parasitoids have already eclosed and teratocytes are released. The results of HPLC analysis indicated the presence of juvenile hormone III together with juvenile hormones I and II in parasitized eggs, but only juvenile hormones I and II in nonparasitized eggs.     

Critical micelle concentrations and stirring are rate limiting in the loss of lipid mass during membrane degradation by phospholipase A2.

In phospholipid membranes attacked by phospholipase A(2) (PLA(2)), accumulation of degradation products influences the binding affinity as well as the catalytic activity of PLA(2). Such accumulation in its turn depends on the rate of membrane degradation and the efflux of degradation products from the membrane, the latter being influenced by the stirring conditions in the system. This complicated process was investigated with a new ellipsometric technique for in situ measurement of membrane mass in a well-defined flow system. Planar phospholipid bilayers were formed on rotating silicon discs in buffer solution. After the addition of 0.05-100 ng/ml of PLA(2) (from Naja mocambique mocambique) to the buffer, mass desorption could be measured with a precision of 3-5 ng/cm(2), that is, about 1% of the surface mass of a single bilayer. Using radiolabeled phospholipids and thin-layer chromatography, it was verified that only the degradation products desorb from the membrane, which was confirmed by the desorption of mixtures of phospholipids, lysophospholipids, and fatty acids. The rotating disc allows the exact calculation of the mass transfer constant for transport-limited exchange of lipid between fluid and disc surface, as a function of rotation rate. By using the mass transfer constant, the critical micelle concentrations, and the mole fractions of products, desorption kinetics could be fully described. The amount of degraded phospholipid could be continuously monitored as the sum of the product mass still present in the membrane, as inferred from the desorption rate, and the mass already lost from the surface. It is concluded that ellipsometry is a suitable tool for studying the effects of PLA(2) on membranes.     

Relative helminth size in crustacean hosts: in vivo determination, and effects of host gender and within-host competition in a copepod infected by a cestode

Crustaceans are important hosts for a number of helminth parasites, and they are increasingly used as models for studying the physiology, ecology and evolution of parasite-host interactions. In ecological studies, this interaction is commonly described only in terms of prevalence and number of larvae per infected host. However, the volume of helminth parasites can vary greatly, and this variation can potentially give important insights into the nature of a parasite-host relationship. It may influence and be influenced, for example, by within-host competition, host size, growth, and life history. Here we present a simple method that allows rapid approximation of the absolute and relative volumes of cestode larvae within copepod hosts of various developmental stages (nauplii, copepodites and adults). The measurements are taken in vivo without much disturbance of the animals, i.e. the technique allows study of growth and development of the parasites in relation to that of their hosts. The principles of this technique can be adopted to other helminth parasites and other crustacean hosts. Using this method in the copepod Macrocyclops albidus infected with the cestode Schistocephalus solidus, we found that the relative parasite size (= `parasite index') ranged from 0.5% to 6.5% of host size 14 days after infection. It was greater in male than in female hosts. With increasing number of parasites per host, the total parasite volume increased while the mean volume of the individual parasites decreased. The magnitude of the observed parasite indices, the large variation that was found within a sample of 46 infected adult copepods, and the observed correlates suggest that this new index can indeed be an important measure of parasite success and its pathogenecity.     

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.     

NMDA receptor dependent and independent components of veratridine toxicity in cultured cerebellar neurons are prevented by nanomolar concentrations of terfenadine

Summary. Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30 min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24 h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate. Terfenadine reduced tetrodotoxin-sensitive inward currents, and reduced intracellular cGMP formation following veratridine exposure. Our data suggest that nanomolar concentrations of TEF may reduce excitatory aminoacid release following neuronal depolarization via a presynaptic mechanism involving voltage sensitive sodium channels, and therefore may be considered as a prototype for therapeutic drugs in the treatment of diseases that involve excitatory aminoacid neurotransmission. Received August 31, 1999 Accepted September 20, 1999     

Low plasma progesterone concentrations are accompanied by reduced luteal blood flow and increased size of the dominant follicle in dairy cows

To investigate the influence of low plasma progesterone (P₄) concentrations on luteal and ovarian follicular development as well as endometrial gene expression in the concomitant and subsequent estrous cycle, 20 lactating dairy (Holstein Friesian and Brown Swiss x Holstein Friesian) cows received either a single treatment with 25 mg prostaglandin F_2α (PGF_2α) on Day 4 Hour 12 (PG1; n = 8), or two treatments (25 mg PGF_2α each) on Day 4 Hours 0 and 12 (PG2; n = 12) of the estrous cycle (Day 1, Hour 0 = ovulation). In four cows, ovulation occurred between 4 and 6 d after the second PGF_2α treatment; these cows and one lame cow were excluded. In the 15 remaining cows with physiological interovulatory intervals (18 to 24 d), P₄, luteal size (LS) and blood flow (LBF), as well as follicular size (FS) and blood flow (FBF), were determined daily until Day 4, immediately prior to (0 h) and 12 h after each PGF_2α treatment, and then every 2 d, from Day 5 to 8 d after the subsequent ovulation. Because P₄ did not differ (P > 0.05) between PG1 and PG2, cows were regrouped according to their mean P₄ concentration from Days 7 to 15, either P₄ <2 ng/mL (P₄L; n = 7) or P₄ >2 ng/mL (P₄H; n = 8). In the treatment cycle, LS was smaller in P₄L than P₄H on Days 13 (P = 0.01) and 15 (P = 0.03), and LBF was lower in P₄L than P₄H on Day 15 (P = 0.02). The dominant follicle of the first follicular wave was larger in P₄L than P₄H on Days 13 (P = 0.03), 15 (P = 0.03), and 17 (P = 0.01). In the subsequent cycle, there were no significant differences between P₄L and P₄H for P₄, FS, LS, and LBF; however, FBF was lower (P = 0.01) in P₄L than P₄H on Day 7. In Group P₄L, endometrial expressions of estrogen receptor α and oxytocin receptor were lower (P = 0.05 and P = 0.03, respectively) at the estrus that preceded treatment compared to the post-treatment estrus. In summary, low P₄ during diestrus was associated with smaller LS, reduced LBF, and larger FS in the treatment cycle, but not in the subsequent cycle.     

Relationship between prevalence of trematode parasite <Emphasis Type="Italic">Diplostomum</Emphasis> sp. and population density of its snail host <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> in lakes and ponds in Finland

Diplostomum sp. is a trematode parasite that infects aquatic snails, e.g. Lymnaea stagnalis (Gastropoda: Lymnaeidae), fish, and fish-eating birds. Ponds and lakes (n = 28) located in Finland between latitudes 61°45′N and 65°30′N were sampled for L. stagnalis, the first intermediate host for Diplostomum sp. L. stagnalis were found in 22 sites out of 28, and Diplostomum sp. in 10 of the 22 snail populations. Among the L. stagnalis populations that were infected by Diplostomum sp., the mean prevalence was 12.8%. Diplostomum sp. occurred in only one out of the seven L. stagnalis populations in four large lakes, but in 9 out of the 16 L. stagnalis populations in small lakes. In the pooled data, a positive correlation (r _s = 0.427; P = 0.047; n = 22) between L. stagnalis density and Diplostomum sp. prevalence was found. The results suggest that Diplostomum sp. is fairly common in L. stagnalis populations in small lakes but rare in large lakes. Furthermore, although trematode parasites, in general, should have a negative effect on snail population density, the study indicates that the relationship between host density and parasite prevalence may greatly differ for individual trematode species, such as Diplostomum sp.     

Activation of protein kinase isoenzymes under near physiological conditions. Evidence that both types (A and B) of cAMP binding sites are involved in the activation of protein kinase by cAMP and 8-N3-cAMP

cAMP-dependent protein kinase I and II (cAKI and cAKII) were incubated under near physiological conditions in the presence of various concentrations of 8-N3-c[3H]AMP or c[3H]AMP. Both types (A and B) of cyclic nucleotide binding sites of cAKI or cAKII were occupied to a similar extent and the degree of their occupation correlated with the degree of kinase activation. cAKI and cAKII bound cAMP in an apparent positively cooperative manner in the presence of Mg2+, ATP. 8-N3-c[3H]AMP dissociated several orders of magnitude faster from site A than site B of the regulatory moiety of cAKII, and was photo-incorporated only when bound to site B.     

Replacement of nitrate by ammonium as the nitrogen source increases the salt sensitivity of pea plants. I. Ion concentrations in roots and leaves

Pea seedlings (Pisum sativum L. cv ‘Kleine Rheinlän-derin’) were grown hydroponically in solutions containing either nitrate (3 or 14 mol m⁻³) or ammonium (3 mol m⁻³) as the nitrogen source. Ammonium nutrition as such had no negative effect on plant biomass production, but drastically increased the sensitivity to moderate salinity (50 mol m⁻³ NaCl). The reasons for this effect are investigated here and in a subsequent paper. The appearance of visible symptoms of salt damage (wilting of marginal leaf areas followed by progressive necrosis) was paralleled by the development of several characteristic modifications in the solute and metabolite contents. Major changes were: (i) high salt (NaCl) accumulation in leaves; (ii) accumulation of ammonium (up to 20 mol m⁻³) and amino acids (up to 110 mol m⁻³) in leaves, but at decreased ammonium uptake rates; and (iii) decreased protein content. In a comparison paper we report on the subcellular distribution of salts, ammonium and metabolites under the above conditions.     

Analysis of changes in congener selectivity during PCB degradation by Burkholderia sp. strain TSN101 with increasing concentrations of PCB and characterization of the bphBCD genes and gene products

We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 μg Kaneclor 300/ml, a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls in medium containing 25 μg Kaneclor 300/ml. Using PCB concentrations of 50–150 μg of Kaneclor 300/ml, the congener selectivity pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 μg Kaneclor 300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2′- and 4′-positions. A 5-kb DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated 99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2,3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the cloned bph gene in biphenyl metabolism. Received: 18 February 1997 / Accepted: 19 August 1997