The effect of infection with Dirofilaria immitis (dog heartworm) on fluid secretion rates in the Malpighian tubules of the mosquitoes Aedes taeniorhynchus and Anopheles quadrimaculatus

Dirofilaria immitis, the parasitic nematode causing the disease dog heartworm, is transmitted by female mosquitoes. During their development, larval nematodes reside in the cells of the Malpighian tubules of these mosquitoes for approx 13 days. We have examined the effect of the presence of these large intracellular parasites on the main physiological function of the Malpighian tubules, i.e. fluid secretion. Rates of fluid secretion were examined in vitro using both normal and infected tubules of the mosquito species Aedes taeniorhynchus and Anopheles quadrimaculatus. Tubules of A. quadrimaculatus show changes in trasport rate during the reproductive cycle. Those of A. taeniorhynchus do not. Infection with larvae of D. immitis had no effect on the rate of fluid secretion in tubules of A. quadrimaculatus. In A. taeniorhynchus by contrast, the tubules show decline in transport with time following infection. The reduction in transport capacity is proportional to the number of worms infecting the tubule. The present paper and separate ultrastructural studies demonstrate that parallel changes in microvillar ultrastructure and epithelial transport rates occur in response to infection by the parasite. In both species examined, the survival of the mosquitoes and their vector potential are determined by factors other than the transport capacities of the infected Malpighian tubules.     

Long-term regulation of sucrose intake by the female mosquito, Aedes taeniorhynchius

Newly emerged female Aedes taeniorhynchus were maintained in cups provided with potometers containing 5, 10, and 25% sucrose solutions. Mortality and the ab lib. intake of sucrose were recorded daily and groups were sampled at regular intervals for analysis of energy reserves.After a week of relatively substantial intake of sucrose at all three concentrations, a constancy in daily intake at much lower levels was observed during the next 4 weeks. The caloric intake was highest in the 25% sucrose solution group. There were no differences in mortality rates at these concentrations. Glycogen (0·7–1·0 cal/female) and triglycerides (3·5dash4 cal/female) reserves reached maximum levels after the first week and stabilized thereafter on all three concentrations. In this species the stabilization in intake of sucrose solution after the first week is correlated with the maximum accumulation of stored energy reserves as opposed to decreased activity or subtle changes associated with senescence.     

Responses of the autonomic nervous system in altitude adapted and high altitude pulmonary oedema subjects

Studies were carried out to ascertain the role of sympatho-parasympathetic responses in the process of adaptation to altitude. The assessment of status of autonomic balance was carried out in a group of 20 young male subjects by recording their resting heart rate, blood pressure, oral temperature, mean skin temperature, extremity temperatures, pupillary diameter, cold pressor response, oxygen consumption, cardioacceleration during orthostasis and urinary excretion of catecholamines; in a thermoneutral laboratory. The same parameters were repeated on day 3 and at weekly intervals for a period of 3 weeks, after exposing them to 3,500 m; and also after return to sea level. At altitude, similar studies were carried out in a group of 10 acclimatized lowlanders, 10 high altitude natives and 6 patients who had recently recovered from high altitude pulmonary oedema. In another phase, similar studies were done in two groups of subjects, one representing 15 subjects who had stayed at altitude (3,500–4,000 m) without any ill effects and the other comprising of 10 subjects who had either suffered from high altitude pulmonary oedema (HAPO) or acute mountain sickness (AMS). The results revealed sympathetic overactivity on acute induction to altitude which showed gradual recovery on prolonged stay, the high altitude natives had preponderance to parasympathetic system. Sympathetic preponderance may not be an essential etiological factor for the causation of maladaptation syndromes.     

In vitro activation of tumoricidal properties in mouse macrophages using the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) incorporated in liposomes

Summary We investigated the ability of free or liposome-incorporated synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) to generate tumoricidal properties in mouse macrophages. As FMLP contains three hydrophobic amino acid residues, it can readily be incorporated into multilamellar vesicles (MLV) consisting of phosphatidylcholine (PC) and phosphatidylserine (PS). The incorporation of FMLP into MLV with a PC:PS ratio of 7:3 mol at FMLP concentrations of up to 10^–4 M did not affect the phagocytosis of liposomes by mouse peritoneal macrophages. Studies with radioactive FMLP revealed that higher levels of FMLP can be delivered to macrophages by liposomes than in the free, nonencapsulated form. Treatment of mouse macrophages with liposome-encapsulated FMLP, but not with free FMLP, generated tumoricidal properties in the macrophages. The mechanism appears to involve an intracellular site since 100-fold concentrations of free FMLP or free N-acetyl-methionyl-leucyl-phenylalanine, the FMLP agonist, failed to competitively inhibit the macrophage's tumoricidal properties generated by liposome-encapsulated FMLP.     

Flight behavior and phase polymorphism in the mosquito Aedes taeniorhynchus

Adults of Aedes taeniorhynchus, reared under two different population densities at 27° under LD 12:12, exhibit differences in their flight behavior. Adults reared under crowded conditions show a main flight peak on the day of emergence as compared to adults reared under non-crowded conditions, which do not. On the basis of their flight activity, these adults are designated as belonging to migrant and non-migrant phases. Adults from both these conditions did not show statistically significant differences in their morphological characteristics, percent glycogen and state of autogeny, but those belonging to migrant phase are much lighter in dry body weight and have lower percentages of lipid than those belonging to non-migrant phase. Analysis of these results and earlier work on this insect suggests that phase polymorphism in this insect is due to the plasticity in its ontogenetic potential.

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Fluggewohnheiten und phasenpolymorphismus bei der mücke Aedes taeniorhynchus

Zusammenfassung Erwachsene Aedes taeniorhynchus, die unter 27° und LD 12:12 bei zwei unterschiedlichen Populationsdichten aufgezogen wurden, weisen Unterschiede in ihrem Flugverhalten auf. Unter Gedrängebedingungen aufgewachsene Imagines zeigen am Schlupftage einen Hauptfluggipfel, nicht gedrängt aufgezogene dagegen nicht. Auf Grund ihrer Flugaktivität werden diese Adulten als Angehörige der migranten und der non-migranten Phase bezeichnet. Die unter diesen verschiedenen Bedingungen aufgezogenen Imagines weisen in ihren morphologischen Eigenschaften, dem prozentualen Glykogengehalt und im Autogeniezustand keine statistisch signifikanten Unterschiede auf, aber die zur Migrantenphase gehörenden haben ein viel geringeres Trockengewicht und geringere Prozentgehalte an Lipiden als die zur nonmigranten Phase zu rechnenden. Die Analyse dieser Ergebnisse und frühere Untersuchungen lassen vermuten, daß der Phasenpolymorphismus bei diesem Insekt eine Folge der Plastizität seines ontogenetischen Potentials ist.

     

Uptake and accumulation of the vital dye hydroethidine in neoplastic cells

Hydroethidine, a reduced form of ethidium bromide, was used as a vital dye in fluorescence assays that allowed visual and semiquantitative monitoring of dye uptake and accumulation by fluorescence microscopy, flow cytometry, image analysis, and microfluorimetry. The excitation and emission filters were chosen to detect hydroethidine and exclude ethidium. Microscopically, there were differences in fluorescence intensities and fluorescence patterns among various tumor cell lines. The fluorescence pattern varied from homogeneous blue in the cytoplasm to blue plus brilliant packets of bluish-white distributed in the cytoplasm. Nuclear staining varied from brown to reddish orange fluorescence. These differences were confirmed by flow cytometry and image analysis. A preliminary survey of various tumors indicated that uptake and accumulation of hydroethidine were dependent on concentration of the dye, duration of cell exposure to the dye, and metabolic state of the cells. Microfluorimetry made possible monitoring of 96 samples in a microculture plate in 30 seconds; thus, this method allows large numbers of samples to be read, with a tremendous savings in time and reagents. The results obtained from the different techniques used were corroborative; therefore, any one of the above techniques may be used in an assay.     

Studies on a natural population of Cyclestheria hislopi (Baird) (Conchostraca: crustacea)

The present paper adds further notes on the biology of Cyclestheria hislopi (Baird). An attempt has been made to study the variation in the natural population of this species based on its birth-rate. C. hislopi has dist inct preference for the plant Hydrilla verticellata (Royle) on which they remain attached. It is considered completely parthenogenic, having no males. The period from October to January can be considered to be the growth period for this species, after which the population declines. This decline has been found to be due to the low birth-rate exhibited during the subsequent months.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

Uptake and resource allocation of inorganic carbon by the temperate seagrasses Posidonia and Amphibolis

Productivity measurements from carbon uptake have been suggested as good indicators of the physiological health of seagrasses. As seagrasses acquire carbon from the surrounding water, the rate of uptake often provide a good measure of the efficiency at which seagrasses meet their resource demands for growth. This rate is often used to assess the photosynthetic efficiency of the plants, a proxy for the physiological status of seagrass. This has special relevance to the Adelaide region as over 5000 ha of seagrasses have been lost from Adelaide coastal waters over the last 70 years, with much of this loss attributed to nutrient inputs from wastewater, industrial and stormwater discharges. This study used an in-situ inorganic carbon isotope-labelling and spike approach to obtain ecologically relevant estimates of seasonal variability in carbon uptake and its allocation in two species of temperate seagrass common to this coast (Amphibolis antarctica and Posidonia angustifolia). Uptake of carbon by the seagrass complex (leaves, roots, phytoplankton and epiphytes) was affected by both season and species. Carbon uptake rates of phytoplankton were generally higher than other components of the system. Uptake rates ranged from 0.01 mg C g^− 1 DW h^− 1 (summer) to 0.61 mg C g^− 1 DW h^− 1 (spring) in Posidonia and 0.02 mg C g^− 1 DW h^− 1 (summer) to 0.93 mg C g^− 1 DW h^− 1 (winter) in Amphibolis. Carbon uptake by the Amphibolis complex was higher than in the Posidonia complex. The Amphibolis complex had higher uptake rates in summer whereas the Posidonia complex was higher in spring. Fine sediments probably from a nearby dredging operation, are likely to have resulted in lower carbon uptake and a reduction in the above-ground and below-ground biomass in summer.