Native Hemolymph Research Articles

Physiological and immunological assessment of two types of crayfish in aquaculture

Due to the need to develop domestic aquaculture of decapod crustaceans, one of the urgent issues is monitoring the health of these aquatic organisms through the most informative bioindicators — hemolymph indicators. The aim of the study was a comparative physiological and immunological assessment of long-toed (Pontastacus leptodactylus) and Australian red-clawed (Cherax quadricarinatus) river crayfish contained in a closed water use facility. The main hydrochemical parameters generally met the existing requirements. The physiological and immunological condition of crayfish was assessed by hematological and cytochemical analyses. Hemolymph samples were taken in vivo by puncture of the ventral sinus. The total number of hemocytes was determined in the native hemolymph in the Goryaev chamber. The number of cells in five large squares was counted. Lysosomal cationic protein was detected in hemocytes by the cytochemical reaction with bromophenol blue, which we previously modified. Red-clawed crayfish have a significantly 45% higher total number of hemocytes, which indicates its greater immune resistance to pathogens compared with long-toed crayfish. The most significant species differences in the hemogram were noted for agranulocytes (HC I) — in long-fingered crayfish, the indicator is higher with a high confidence coefficient (t). With a high degree of confidence, it is possible to judge a higher proportion of semigranulocytes (HC II) in red-lobed crayfish. The proportion of HC IV of long-fingered crayfish is greater than that of red-clawed crayfish. In red-clawed crayfish, the average cytochemical coefficient of ACC is lower than in long-fingered crayfish. This also confirms its high immune resistance due to the consumption of highly cytotoxic cationic protein in the process of immune protection.

EDTA-Na<sub>2</sub> - anticoagulant for hemolymph of australian red claw crayfish (cherax quadricarinatus)

Purpose: selection of anticoagulants based on EDTA-Na2 and methods of their use for working with the hemolymph of the Australian red claw crayfish (C. quadricarinatus)Materials and methods. For the study, different-sized males and females of the australian red-clawed crayfish (Cherax quadricarinatus (Von Martens, 1868)) were used. EDTA-Na2 was used as an anticoagulant, the concentrationof which was 4%. A 2 ml syringe with a 23G needle for hemolymph removal was pre-washed with anticoagulant remaining in the needle and the needle cone (about 1/3 of the volume of the needle cone was filled with a solution). The experiment is presented in the following series: 1. differences between the total hemocytes number (THC) and the proportion of granulocytes in native hemolymph and treated with a small amount of EDTA-Na2; 2. differences in the content of total hemolymph protein (THP) in native and treated with a small amount of EDTA-Na2 hemolymph; 3. changes in THC and the proportion of granulocytes in hemolymph treated with a small amount the amount of EDTA-Na2 immediately after sampling and a day later. To carry out the work, samples of 20 individuals were used, while for the first two series, two samples of hemolymph were taken from each, and one from the third. Syringes with hemolymph treated with anticoagulant were stored in the refrigerator at a temperature of 8.5 °C. Additionally, similar studies were carried out on differences in the THC and proportion of granulocytes in the hemolymph immediately after sampling with treatment with a multicomponent anticoagulant consisting of 4 g of EDTA-Na2, 3 g of sodium citrate, 2 g of glucose and 1 g of NaCl per 100 ml of distilled water. Hemolymph was taken with a syringe from the ventral sinus. Three indicators were determined: the total number of hemocytes (THC), the proportion of granulocytes and the total protein content (THC). THC and proportion of granulocytes was determined in the Goryaev chamber under a light microscope. THP was determined by the refractometric method.Results. The study revealed significant differences in the proportion of granulocytes (p<0.05), which are 32% more in the anticoagulant treated than in the native hemolymph, which can be explained by the uniform distribution of all types of hemocytes in the sample. There were no significant differences in the remaining indicators of all series. Studies of hemolymph treated with a multicomponent anticoagulant showed a low level of hemocyte preservation (p<0.05) and the proportion of granulocytes compared to the indicators immediately after selection, therefore, they are not reflected in the work. Conclusion. The data obtained indicate that there is a possibility of using four percent EDTA-Na2 when working with hemolymph. The anticoagulant prevents the formation of gel and the rapid destruction of hemocytes, promotes the uniform distribution of cells in the Goryaev chamber and allows the use of a refractometer in determining the total blood protein. The use of an anticoagulant contributes to the preservation of hemocytes during the day, at a level that allows the use of EDTA-Na2 in practice. At the same time, work should continue on the development of methods for storing and transporting blood treated with an anticoagulant.

The Effect of Temperature on the pH, d-Lactate and Haemocyanin Oxygen Affinity ofOpistophthalmus latimanus(Koch) (Scorpionidae) Haemolymph

The pH and d-lactate of the haemolymph from the scorpion, Opistophthalmus latimanus, were measured at different ambient temperatures. The oxygen affinity and molecular cooperativities of haemocyanin in its native and dialysed form were determined when exposed to different ambient temperatures. D-lactate concentration in native haemolymph increased significantly from 0.31 mmol/l at 7 °C to 1.93 mmol/l when haemolymph was exposed to 25 °C for 6 hours but decreases to 0.32 mmol/l at 37 °C The extinction coefficients of O. latimanus haemocyanin at 240 nm and 278 nm were 2.41 and 11.26, respectively, and correspond well with similar data on the crustacean Limulus polyphemus. The subunit cooperativity (n50) of the native haemocyanin molecule increased linearly from n = 3.00 to n = 5.34 at 37 °C. A d-lactate effect on n50 between pH 7.40 and 7.87 could be demonstrated while the heat of oxygenation (ΔH) was -127.64 kJ/mol between 17 °C and 25 °C Like other scorpions investigated, haemocyanin of O. latimanus exposed to wide temperature regimes show a strong temperature dependence of oxygen affinity (as P50) and cooperativity (as n50).

Structural study of Carcinus maenas hemocyanin by native ESI‐MS: Interaction with L‐lactate and divalent cations

The interaction of L-lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry under conditions preserving noncovalent interactions (native ESI-MS). C. maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L-lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamer-specific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L-lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations.

The archaeogastropod molluscHaliotis iris: tissue and blood metabolites and allosteric regulation of haemocyanin function

We investigated divalent cation and anaerobic end-product concentrations and the interactive effects of these substances and pH on haemocyanin oxygen-binding (Hc-O(2)) in the New Zealand abalone Haliotis iris. During 24 h of environmental hypoxia (emersion), D-lactate and tauropine accumulated in the foot and shell adductor muscles and in the haemolymph of the aorta, the pedal sinus and adductor muscle lacunae, whereas L-lactate was not detected. Intramuscular and haemolymph D-lactate concentrations were similar, but tauropine accumulated to much higher levels in muscle tissues. Repeated disturbance and short-term exposure to air over 3 h induced no accumulation of D- or L-lactate and no change in [Ca(2+)], [Mg(2+)], pH and O(2)-binding properties of the native haemolymph. The haemolymph showed a low Hc-O(2) affinity, a large reverse Bohr effect and marked cooperativity. Dialysis increased Hc-O(2) affinity, obliterated cooperativity and decreased the pH-sensitivity of O(2) binding. Replacing Mg(2+) and Ca(2+) restored the native O(2)-binding properties and the reverse Bohr shift. L- and D-lactate exerted minor modulatory effects on O(2)-affinity. At in vivo concentrations of Mg(2+) and Ca(2+), the cooperativity is dependent largely on Mg(2+), which modulates the O(2) association equilibrium constants of both the high-affinity (K(R)) and the low-affinity (K(T)) states (increasing and decreasing, respectively). This allosteric mechanism contrasts with that encountered in other haemocyanins and haemoglobins. The functional properties of H. iris haemocyanin suggest that high rates of O(2) delivery to the tissues are not a priority but are consistent with the provision of a large O(2) reserve for facultatively anaerobic tissues during internal hypoxia associated with clamping to the substratum.

Modulators of haemocyanin oxygen affinity in the hypoxia- and sulphide-tolerant Baltic isopod Saduria entomon (L.).

Dialysed haemocyanin from the isopod Saduria entomon had a considerably increased oxygen affinity (lower P50) and Bohr factor (-1.71) compared to native haemocyanin (Bohr factor -1.36) indicating that dialysis removes a small molecule size modulating factor decreasing the affinity of native haemolymph. Dialysed haemocyanin had a slightly lower co-operativity (2.42 +/- 0.3) than native haemocyanin (2.9 +/- 0.2). L-Lactate (10 mmol l(-1)) improved oxygen affinity by 1-1.5 torr while urate had no effect. Mg2+ affected affinity in a pH-dependent manner (Bohr-factor increased to -1.67) while Ca2+ had no effect on the Bohr factor but increased affinity with ca 1 torr. Thiosulphate changed the Bohr factor to -1.75 to -1.82, similar to dialysed blood. Co-operativity was in neither case affected. The haemocyanin characteristics of S. entomon are similar to those of crustaceans from hydrothermal vents. These characteristics are probably general for crustaceans that are more or less permanently exposed to sulphide.

Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.

Oxygen transport by the hemocyanin of the protobranch mollusc Solemya reidi

Oxygen transport by the hemocyanin of the protobranch mollusc Solemya reidi Bernard was studied in native hemolymph samples. Clams were collected from two different reducing environments, beneath log booms in Alberni Inlet, British Columbia, Canada, and from the sewage effulent in Santa Monica Bay, California, USA. The hemocyanin concentration in a pooled hemolymph sample (n = 10 individuals) was 33.5 mg ml−1. The mean hemolymph pH of five Alberni clams maintained for 3 wk in mud was 7.96 ± 0.06. No significant variation in hemocyanin oxygen-affinity or cooperativity was found for hemocyanin in whole hemolymph samples from these five individually studied clams. There was a significant difference only at 15 °C in the oxygen affinity of hemocyanin in pooled whole-hemolymph samples from S. reidi collected from Alberni Inlet compared with clams collected from Santa Monica Bay. Little effect of temperature on hemocyanin oxygen-affinity was found for temperatures below 20 °C; above 20 °C, the oxygen affinity was reduced for clams from both sites. Temperature and pH had no apparent affect on hemocyanin cooperativity. Moderate Bohr shifts were found at all temperatures examined. The presence of physiologically relevant concentrations of thiosulphate in hemocyanin samples resulted in a decrease in hemocyanin oxygen-affinity, opposite to the effect on hemocyanin found for the hydrothermal vent crab Byth-ograea thermydron, but thiosulphate had no effect on hemocyanin cooperativity.

Hemocyanin oxygen-binding properties of a deep-sea hydrothermal vent shrimp: Evidence for a novel cofactor

Rimicaris exoculata is a caridean shrimp from the family Alvinocarididae which forms the dominant species around deep-sea hydrothermal vents from the Mid-Atlantic Ridge (MAR). Seeking respiratory adaptations to the hydrothermal environment, we have analysed the oxygen-binding properties of Rimicaris hemocyanin (Hc) in relation with temperature, pH, and lactate variations. Rimicaris native Hc is mostly composed of hexamers. It showed a high oxygen affinity (P50 approximately 3 Torr at pH 7.5, 15°C), a large Bohr effect (ΔlogP50/ΔpH = −1.87 ± 0.25, n = 6), a moderate lactate effect (ΔP50/Δlog[lac] = −0.12) and almost no temperature effect (ΔH = −1.23 kJ.mol−1 15–35°C). Most surprisingly, dialysis of native hemolymph elicited a large increase of HC-O2 affinity, an effect opposite to the usual trend observed for crustacean Hcs. Moreover, this increase in affinity could be reversed by adding an ultrafiltrate of native hemolymph to a dialysed sample, thus unveiling the existence of a dialysable yet unknown cofactor which decreases Hc-oxygen affinity. J. Exp. Zool. 277:357–364, 1997. © 1997 Wiley-Liss, Inc.

Steady-state potentials in ovarian follicles of a moth, Hyalophora cecropia

Ion substitutions and ion-selective microelectrodes were used to study the steady state potential ( E m) between the oöcyte and the medium surrounding ovarian follicles in Hyalophora cecropia, a system developing in a high K + low Na + haemolymph. Potassium channels were affected by the osmolarity of the medium, being maximally open at 300 mOsm, the osmolarity of the native haemolymph. In a 300 mOsm medium, E m changed 26 mV/decade change in [K +] 0, and was slightly positive to E k (1.9 mV; P = 0.035). In most follicles, E m did not vary with changes in [Cl −] 0. Follicles with small E m (−20 to −30 mV), by contrast, included some that depolarized on removal of external Cl −; after several minutes, these follicles subsequently hyperpolarized spontaneously and at the same time adopted the more common Cl − impermeable state. Under physiological conditions, with K + channels open and Cl − permeability low, protons also contributed to E m, which changed at a rate of 14 mV/pH unit change; when K + channels were blocked with tetraethylammonium/Ba 2+, this slope increased to >40 mV/pH unit. Since the cytosolic pH of follicles incubated in the standard pH 6.5 incubation medium averaged 7.3, E H is very positive to E m. Calculations from chord conductance theory indicated that the measured E m would be achieved if g k = 49· g H. Together, H + and K + accounted for up to 69% of the measured steady state potential; chilling the cells mimicked previously reported treatment with azide or vanadate, and confirmed that one-third to one-half of the steady state potential is metabolically maintained.

Oxygen consumption and haemocyanin function in the freshwater snail Marisa cornuarietis (L.)

1. 1. The MO 2 for branchial respiration in adult snails increased from 0.24 mmol/l/O 2 kg/hr at 18°C to 0.83 mmol/l/O 2 kg/hr at 40°C. Q 10 values were 2.75 between 35 and 40°C and 1.8 between 18 and 30°C. 2. 2. The haemocyanin (31.9 ± 5.8 mg/ml) has a high oxygen affinity (6.28 ± 0.8 at 25°C) with a reversed Bohr effect measured between a pH of 6.80 and 7.95 with gelchromatographed haemolymph, and measured between a pH of 7.34 and 8.10 for native haemolymph. 3. 3. Growth rate is optimal between 27 and 30°C whilst at 24°C stunted growth was found. 4. 4. At 25°C the same MO 2 values were found for aerial and aquatic respiration.

Antibacterial activity in the hemolymph of myriapods (Arthropoda)

The hemolymphs of two diplopod ( Chicobolus sp. and Rhapidostreptus virgator) and two chilopod species ( Lithobius forficatus and Scolopendra cingulata) were tested for the presence of antibacterial substances using Petri dish tests. The native hemolymph of all species had substances acting on living Micrococcus luteus, whereas only Rhapidostreptus, Scolopendra, and Lithobius were effective against lyophilized Micrococcus. The antibacterial activity against living Micrococcus increased after inoculation with bacteria ( Enterobacter cloacae β-12) in Chicobolus and Rhapidostreptus and also against lyophilized Micrococcus in the latter. Thus, these effects appear to be inducible. None of the myriapods tested had any bacteriostatic effect on Escherichia coli D-31 whereas the growth of gram-negative E. cloacae was inhibited. The antibacterial substances in the diplopod species were unstable when heated but were resistent to freezing. At least two antibacterial substances (a lysozyme-like one and another substance) are considered to occur in Myriapoda.

A strategy for solubilizing delipidated apolipoprotein with lysophosphatidylcholine and reconstitution with phosphatidylcholine

The apolipoproteins of insect lipophorin were dissociated in guanidinium chloride and isolated by gel permeation chromatography. Over 98% of the total lipid in lipophorin was associated with apolipophorin I (apoLp-I), thus suggesting this apolipoprotein to be the lipid binding component of the particle. ApoLp-I was delipidated with ethanol/ether and solubilized in buffer that contained radioactive lysophosphatidylcholine ([3H]LPC) above the critical micellar concentration. Sonic irradiation of radioactive phosphatidylcholine ([14C]PC) with [3H]LPC-solubilized apoLp-I at a molar ratio of 318 resulted in reconstituted lipophorin (RLp-I). [3H]LPC was bound to fatty acid free bovine serum albumin and was separated from RLp-I by density gradient ultracentrifugation and gel permeation chromatography. Negatively stained RLp-I particles were quasispherical with an average radium of 55 A, and their overall morphology and secondary structure were similar to those of native hemolymph lipophorin. The RLp-I particle had a rho = 1.137 g/mL, a Mr approximately 5.2 X 10(5), and a [14C]PC:apoLp-I molar ratio of 308. From the compositional analysis, molecular size, trypsinization, and lipolysis with phospholipase A2, we concluded that each RLp-I particle contained one molecule of apoLp-I and a monomolecular layer of [14C]PC. When injected into the hemolymph of adult moths in vivo, RLp-I was loaded with lipid, as judged by a decrease in its density both in the presence and in the absence of adipokinetic hormone. The similarities in morphology and immunology of RLp-I and native lipophorin, together with the ability of RLp-I to load lipid, suggest that reconstituted lipophorins may serve as models to probe lipophorin structure and function.

Effects of Freezing on the Function and Association State of Crustacean Haemocyanins

The addition of protease inhibitors and antibiotics provides a valuable means of stabilizing proteins in solution but this is less desirable if the physiological properties of a biological fluid, such as haemolymph, are to be accurately evaluated. Freezing haemolymph has proved to be a convenient way both of transporting samples from remote sites and of storage for longer periods. Although many investigators have suspected and intimated that freezing may affect haemocyanin function there are few data describing exactly what these effects may be. Mangum (1983a) provided some data for Squilla empusa haemocyanin indicating that freezing did not alter the P50 affinity but did reduce the cooperativity of O2 binding. Similar results were reported for Palaemon elegans (Bridges et al. 1984; Morris et al. 1985). These reports that cooperativity may be reduced by freezing indicate that structural changes occur that may lead to changes in affinity, despite the P50 remaining unchanged. The haemocyanins from two macruran and two brachyuran species were investigated; Homarus vulgaris (Selbie) and Cancer pagurus (L.) from Europe, together with Hemigrapsus nudus (Dana) and Cambarus diogenes (Girard) from North America. The effect of freezing on the affinity and cooperativity of oxygen binding was determined by the construction of oxygen equilibrium curves for fresh, native haemolymph and for the same haemolymph subjected to a known number of freezing cycles. The freezing protocol consisted of flash freezing (—80°C) the haemolymph in small samples which were kept frozen for 30 min before thawing at room temperature. Any required sample was withdrawn and the freezing cycle repeated appropriately. Oxygen equilibrium curves were constructed at 15 °C as described by Bridges et al. (1984) or Morris et al. (1988). Both methods are spectrophotometric and directly comparable. Oxygen affinity was analysed as the Pso in dependence of pH and the cooperativity was calculated for the same equilibrium curves (Fig. 1). Using a fiducial limit of P = 0-05, covariance analysis showed no significant change in AlogPso/ApH with respect to freezing. None of the four haemocyanins exhibited significant dependence of cooperativity (n^) on

Ca2+-dependent proteolytic activity in crab claw muscle. Effects of inhibitors and specificity for myofibrillar proteins.

The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. We describe here the role of Ca2+-dependent proteinases in the turnover of myofibrillar protein in normal anecdysial (intermolt) claw muscle. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 X g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The Ca2+-dependent proteinase is a sarcoplasmic cysteine proteinase that shows maximal activation at 1 mM Ca2+ at neutral pH. Since approximately 28% of the activity remains at 1.5 microM Ca2+, the enzyme is partially active at physiological Ca2+ concentrations. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: 1) heavy chain (Mr = 200,000) is cleaved into four large fragments (Mr = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (Mr = 18,000) is cleaved to a 15,000-Da fragment; 2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.

A method for quantitative determination of ice nucleating agents in insect hemolymph.

The relationship between the concentration of insect hemolymph ice nucleators in samples of 0.9% NaCl solution and the supercooling points of the samples was determined by using a dilution technique. The supercooling points were only moderately reduced following dilution by a factor of up to 103, whereas dilution beyond this point caused a marked drop in the supercooling points. The dilution factor corresponding to a 50% reduction in the nucleating activity of native hemolymph is taken as a measure of the concentration of ice nucleators in native hemolymph. This method was used to determine the concentration of ice nucleators in the hemolymph of Eurosta solidaginis larvae from Minnesota and Texas, acclimated to different temperatures. Significant levels of nucleators were found only in larvae from Minnesota, and +5 °C was found to be the optimal temperature for nucleator formation. This comparatively high temperature optimum is interpreted as a physiological adaptation, ensuring sufficient nucleator levels in the hemolymph by the time of the first exposure to freezing temperatures in the winter.

Immunochemical analysis of hemolymph clotting in the insectLeucophaea maderae (Blattaria)

Hemolymph clotting in the Blattarian speciesLeucophaea maderae was analyzed by immunological methods. 1. Experimentally induced (by freezing and thawing) disintegration of hemocytes — in the absence of plasma proteins — is followed by the formation of a clot-like gel. This gel resists treatment with strong detergents, but is dissolved by reducing agents and proteolytic enzymes. The gel material forms part of the native hemolymph clot. It is mainly contained in the granules of the hemocytes from which it is released during clotting. This hemocyte derived clotting protein is calledhemocyte coagulogen. 2. During normal hemolymph clotting one of the plasma proteins disappears from the hemolymph; this protein also becomes incorporated into the native hemolymph clot which is — as compared to the gel — more rigid and not soluble in reducing agents. This second clotting protein — theplasma coagulogen —cannot be found in intact hemocytes. 3. Clotting of the plasma coagulogen is possible only in the presence of the hemocyte coagulogen. 4. Both coagulogens are shown to be present in theislands of coagulation at exactly the same places. 5. Conditions of plasma clotting, differences in solubility between gel and clot, and the common occurrence of both coagulogens in the islands of coagulation are strong arguments for an interaction of the two coagulogens during clotting; the hypothesis is put forward that the two coagulogens in the native clot are interconnected to form a common network.

Studies on the synthesis of lipoproteins during larval-pupal development of Hyalophora cecropia

The synthesis of haemolymph lipoproteins at 3 developmental stages during larval-pupal development of the silkworm, Hyalophora cecropia, has been studied. The haemolymph lipoprotein concentration is very low in the fourth-instar larva, but increases sharply in the fifth-instar larva and reaches a maximum in the pharate pupa. Fat body from fifth-instar larvae, pharate pupae, and newly ecdysed pupae incorporated labelled lipid and protein precursors into lipoproteins, and when such fat body was incubated with haemolymph labelled lipoproteins were recovered from the haemolymph. Puromycin (2 mg. per ml.), when added to the incubation medium, partially inhibited the incorporation of labelled amino-acids into the protein moiety of the lipoproteins, but the incorporation of labelled fatty acids into the lipid moiety was unaffected. Fat body extracts (from which the lipids had been removed) incorporated labelled haemolymph lipids into lipoproteins when incubated in vitro. Radioassay of the lipid fractions from these lipoproteins assembled in vitro revealed a similarity to those of the native haemolymph lipoproteins after incubation with prelabelled fat body. From the data it is suggested that the fat body may contain apo-lipoproteins that are able to bind with haemolymph lipids to form lipoproteins.

The haemocyanin of Pila leopoldvillensis—II. Quaternary structure and oxygenation

1. 1. Pila leopoldvillensis haemocyanin has been investigated under different pH conditions by means of combined electron microscopy and sedimentation analysis. 2. 2. The shape of the molecule (a six-layered hollow cylinder, dia. 345 Å, height 385 Å), its dissociation products into halves and tenths and the reassociation products have been described, as well as the shape of the molecules in the native haemolymph. 3. 3. A preliminary study of the oxygenation has indicated an oxygen-binding capacity of 0·34 and a normal Bohr effect.