Heterosynthetic origin of the major yolk protein,vitellin, in a nereid,Perinereis cultrifera (polychaete annelid)

• 1.1. An examination of proteins synthesized by Perinereis cultrifera oocytes incubated in vitro with [³H]leucine clearly shows that these cells are not capable of synthesizing the main yolk protein previously identified in this worm.
• 2.2. In addition, the detection of radiolabelled vitellin in oocytes after in vitro incubation of an oocyte-coelomocyte cell mixture in presence of [³H]leucine strongly suggests that the coelomocytes, free cells in the coelomic cavity, synthesize and secrete a vitellin precursor, vitellogenin, that is subsequently taken up by the oocytes.
• 3.3. Two native proteins differing in mol. wt but reacting with anti-vitellin antibodies have been identified in coelomocyte incubation medium. Also found in the coelomic fluid, they have been designated VG1 (M_r = 530,000) and VG2 (M_r = 320,000).
• 4.4. The two vitellogenins consist of a single type of polypeptide of M_r = 176,000 and are incorporated in the oocytes where they are apparently observed under a single molecular form corresponding to VG1, the highest mol. wt protein similar in size to the initial form of vitellin (VI, 530,000).
• 5.5. From these data, it seems likely that VG2 is a monomeric molecule that is taken up by the oocytes as a dimer of VG1.
• 6.6. We conclude that P. cultrifera accumulates vitellin heterosynthetically and that vitellogenin is produced by the coelomocytes. Moreover, a single polypeptide similar in size to the polypeptidic component of secreted vitellogenin has been detected in the coelomocytes.
• 7.7. Since this polypeptide has been identified previously as the single intraoocytic precursor of the four lower mol. wt products that make up the mature form of vitellin (V5), it appears that P. cultrifera exhibits for vitellogenin a processing pathway in which cleavage of the precursor occurs only after uptake by the oocyte.

     

Comparative vitellin pattern and cladogenesis in Bacillus (insecta: Phasmatodea)

• 1.1. Under denaturing conditions (SDS-PAGE) the two natural vitellins of Bacillus taxa released five different polypeptides (A₁, A₂, A₃, B₁, B₂).
• 2.2. A₂ and B₂ bands from the two bisexual species (B. rossius and B. grandii) were found to differ; furthermore a non-vitellin yolk protein characterizes the subsepecies B.g. benazzii.
• 3.3. From gels and their densitometric scanning profiles it is clear that parental polypeptides are expressed in the thelytokous parthenogenetic hybrids (B. whitei, B. lynceorum) and in the hybridogenetic B. rossius-grandii benazzii.
• 4.4. A comparative approach of vitellin patterns appears fully adequate for tracing phylogenetic relationships and recognizing cladogenetic events.

     

Multiplicity of dog kidney high-Km aldose reductase and conversion mechanism into aldose reductase

• 1.1. High-K_m, aldose reductase purified from dog kidney inner medulla was easily converted into aldose reductase by incubation in the neutral buffer solution.
• 2.2. High-K_m, aldose reductase was found to be in multiple forms, and was separated into three kinds of species designated as a-, b- and c-forms by HPLC.
• 3.3. The a-form observed as a single peak by HPLC was assumed to be present in three forms (al-, a2- and a3-forms), one was aldose reductase (a 1-form) and the others were the precursors of aldose reductase (a2- and a3-form).
• 4.4. The b-form was rapidly converted into the a3-form, followed slowly by the a2-form and finally into the a 1-form.
• 5.5. The c-form was either directly converted into the al-form, or indirectly into the a2-form followed by the al-form.
• 6.6. Four kinds of species (a2-, a3-, b- and c-forms) of high-Ap, aldose reductase were finally converted into aldose reductase (al-form).

     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Evidence for a molecular polymorphis of cholinesterase in Sepia officinalis (cephalopoda: decapoda)

• 1.1. Three forms of cholinesterase were sequentially extracted from head and tentacles of Sepia officinalis and noted as low-salt (LSS), detergent (DS) and high-salt (HSS) soluble. They represent about 24, 30 and 46% of total activity.
• 2.2. All enzyme forms seem to be amphiphilic proteins with hydrophobic domains interacting with non-ionic detergent (Triton X-100) and giving self-aggregation (LSS form).
• 3.3. The DS form is membrane-anchored by a phosphatidylinositol, while the HSS form is likely linked to some proteoglycan molecule of the extracellular matrix by ionic interactions.
• 4.4. According to V_max/K_m values, all the enzymes are acetylcholinesterases, even if hydrolyze propionylthiocoline at the highest rate.
• 5.5. Some kinetic and molecular properties of the studied enzymes are compared with those of other cholinesterases from vertebrates and invertebrates. Possible phylogenic and adaptive features are discussed.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

Reabsorption and accumulation of α-amino-iso-butyric acid in the nephridia of Sabella pavonina Sa vigny (Annelida polychaeta)

• 1.l. High amino acid concentrations were found in the anterior coelomic fluid of a Polychaeta (Sabella pavonina Savigny).
• 2.2. The concentrations being much higher in the fluid which penetrates the nephrostomia into the nephridia lumen than in the final urine indicates that the nephridia reabsorbs large amounts of amino acids.
• 3.3. Nephridial perfusion experiments showed that an amino acid analogue (α-amino-iso-butyric acid, AIB) is transported by the nephidia.
• 4.4. The transport took place across the nephridial wall owing to the presence of a carrier-mediated transport system and a diffusion system.
• 5.5. For the carrier-mediated transport, the V_max was 0.234 ± 0.025 nmol·min⁻ and the K_m 3.715 ± 0.315mmol·l⁻.
• 6.6. AIB accumulated in the nephridial cells up to a maximum rate of 01.17 nmol·min⁻.
• 7.7. Intracellular accumulation stopped increasing when the V_max for reabsorption was reached.
• 8.8. These results indicate that the carrier-mediated transport of AIB is located at the apical membrane of the nephridial cell, and that AIB transport by simple diffusion takes place through the paracellular pathway.

     

Decarboxylation of uroporphyrinogen I and III in 2,3,7,8-tetrachlorodibenzo-p-dioxin induced porphyria in mice

• 1.1. The decarboxylation of uroporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in mouse liver supernatant was compared in relation to substrate concentrations.
• 2.2. In this species uroporphyrinogen III was the best substrate judging by the criteria of K_m/V_max (estimated for total porphyrinogens) and was converted into coproporphyrinogen faster than its series I isomer.
• 3.3. The difference between the two isomers was mainly due to the first decarboxylation.
• 4.4. This difference was confirmed by calculation of the Hill coefficient and of Lineweaver-Burk plot which suggested that isomer I induced negative cooperativity in the active centre of the enzyme.
• 5.5. After treatment with a porphyrogenic dose of TCDD (25 μg/kg/week for 9 weeks) differences between uroporphyrinogen I and III as substrate were maintained.
• 6.6. In addition treatment reduced V_max and K_m (estimated for total porphyrinogens) of liver porphyrinogen carboxy-lyase to about half control values for both isomers.
• 7.7. V_max was reduced mainly because of the formation of smaller amounts of all products of decarboxylation, and K_m because more heptaporphyrinogen was formed than coproporphyrinogen.
• 8.8. Values of the Hill coefficient and Lineweaver-Burk plots suggested TCDD induced altered substrate affinity for isomer III too.
• 9.9. Treatment with TCDD did not affect the decarboxylation of uroporphyrinogen III by RBC porphyrinogen carboxy-lyase, estimated from K_m and V_max for total porphyrinogens formed.

     

Factors affecting oxygen consumption in wild-caught yellow-bellied marmots (Marmota flaviventris)

• 1.1. All age groups gained mass during the active season, but mass-gain of adult females was delayed during lactation.
• 2.2. The relationship of body mass to metabolic rate varied widely; when the relationship was significant, R² varied from 10.3 to 72.6%. Body mass affects V_O2 more during lactation than at any other period.
• 3.3. Mean VinO₂ of adult males was higher in June than that of adult, non-lactating females.
• 4.4. V_O2 of reproductive females was significantly higher during lactation than during gestation or postlactation because specific V_O2 varied. Specific V_O2 of non-reproductive females declined over the active season.
• 5.5. Specific V_O2 of all age groups declined between the premolt and postmolt periods. The reduced maintenance costs can contribute 20–46% to daily growth.
• 6.6. Observed V_O2 was lower than the value predicted from intraspecific or interspecific Bm:M regressions.
• 7.7. V_O2 of wild-caught marmots was lower than that of marmots maintained in the laboratory, probably because of dietary differences.
• 8.8. Because basal metabolism is a stage on a food-deprivation curve, we suggest that basal metabolic rate is not an appropriate measure of the metabolic activity of free-ranging animals.

     

Isolation and partial characterization of vitellin from the egg of the giant tiger prawn,penaeus monodon

• 1.1. The results obtained from the present study show that vitellin (yolk protein), a glycolipoprotein derived from the giant tiger prawn, Penaeus monodon, consists of four polypeptides, Ep1, Ep2, Ep3 and Ep4, with molecular weights of 168, 104, 83 and 74 kDa, respectively. This protein was purified using SDS-PAGE and gel-elution.
• 2.2. Antisera against Ep2, Ep3 and Ep4 fractions showed very strong specific binding reactivity when reacted with shrimp ovaries at developing stages.
• 3.3. No crossreaction was observed among Ep2, Ep3 and Ep4 fractions of vitellin. However, anti-Ep2 and anti-Ep3 were demonstrated to be able to react with Ep1 fraction. The homogenates obtained from ripe ovaries and hemolymph of vitllogenesis females showed an immuno-identical pattern to those obtained from egg extracts.
• 4.4. The proteolysis mapping for each fraction showed that Ep1, the high molecular weight fraction, contained Ep2 and Ep3 fragments. These results suggest that Ep1 may be a precursor for Ep2 and Ep3.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

Effect of experimental ventilation of the skin on cutaneous oxygen uptake in the carp,Cyprinus carpio

• 1.1. Cutaneous O₂ uptake in the carp, Cyprinus carpio, was determined at various water flow rates across the skin (.V) ranging from 2.5 to 40 ml/min, using flow-through respirometers.
• 2.2. When thickness of water flow was 2mm, cutaneous O₂ uptake remained stable (about 3.8 nmol/cm²/min) at a .V of 20–40 ml/min and decreased with .V below 20 ml/min.
• 3.3. When thickness of water flow was 4 mm, cutaneous O₂ uptake decreased with .V below 40 ml/min.
• 4.4. Apparent water velocity (U') was calculated dividing .V by an area of a cross section of the water flow (0.5 and 1.0 cm² respectively). In both experiments, cutaneous O₂ uptake decreased with U' below 0.7 cm/sec.
• 5.5. This suggests that cutaneous O₂ uptake in the carp is limited at a low water velocity by a resistance of the hypoxic boundary layer.

     

Purification of endo- and exolaminarinase and partial characterization of the exoacting form from the copepod acartia clausi (Giesbrecht, 1889)

• 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
• 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
• 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
• 4.4. The kinetic parameters V_m and K_m vary with the temperature; the affinity constant (K_a) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
• 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.

     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

Modification of glyceraldehyde-3-phosphate dehydrogenase,EC 1.2.1.12, isolated from rainbow trout during acclimation at 5 or 15°C—I. Changes in the activity of individual muscle isolates as evidence for variable adaptive responses

• 1.1. The mean K_m and V_max values for G3PDH isolated from the lateral muscle of cold-adapted (5°C) rainbow trout, Salmo gairdneri, were twice those of enzyme from warm-adapted (15°C) trout when assayed at 7°C but not at any other temperature.
• 2.2. The entropy of activation of warm enzyme was about 3 times that of cold enzyme. However, enthalpy or free energy of activation among acclimation groups differed less or not at all.
• 3.3. Individual G3PDH isolates within either adaptation group differed in kinetic characteristics.

     

Preliminary studies on the characteristics of phenylalanine and β-methyl-glucoside transport in the tench intestine in vitro

• 1.1. The unidirectional transepithelial fluxes of L-phenylalanine, β-methyl-D-glucoside and sodium ions across emusculated sheets of tench mid-intestine were determined in flux chambers.
• 2.2. No net sodium flux was detectable, but phenylalanine was preferentially transferred from the mucosal to the serosal fluid.
• 3.3. There was also a net movement of β-methyl-glucoside towards the serosal medium, but it was much smaller than that of phenylalanine.
• 4.4. This transport was accompanied by an accumulation of each substrate from the mucosal medium into the tissue to a similar level and against a concentration gradient.
• 5.5. The poor transfer of the monosaccharide into the serosal medium could therefore be attributed to a low permeability of the baso-lateral membrane of the enterocyte for this substance.
• 6.6. The influx of L-phenylalanine and of β-methyl-d-glucoside into the epithelial cells of tench midintestine was examined by incubating slices of emusculated intestine in radioactively-labelled solutions of the substrate for 2 min.
• 7.7. The steady-state uptake was assessed after similar incubations lasting 45 min.
• 8.8. Phenylalanine influx obeys the Michaelis-Menten equation with a K_m of 2.9 mM and is dependent on the presence of sodium ions in the incubation medium.
• 9.9. β-Methyl-glucoside influx reveals the same characteristics with a K_m of 2.0 mM but a considerably lower V_max; in addition, it is inhibited by galactose.
• 10.10. The influx of both substrates is reduced by harmaline, which also inhibits the uptake of radioactive sodium by this preparation.
• 11.11. The steady-state uptake of β-methyl-glucoside is also inhibited by ouabain and by 2.4-dinitrophenol.
• 12.12. These results suggest that the mechanisms for sodium-dependent influx of monosaccharides and neutral amino-acids in the tench intestine are similar to those found in mammalian tissues.
• 13.13. The principal difference appears to involve the release of monosaccharides across the baso-lateral membrane of the enterocyte.

     

Amino acid accumulation in kidney cortex of lambs and piglets as affected by insulin

• 1.1. Insulin stimulated intracellular accumulation of α-amino-isobutyric acid (AIB) in kidney cortex slices from young lambs and piglets.
• 2.2. The effect was similar in the absence or presence of glucose.
• 3.3. The induction of the stimulatory effect on renal AIB transport was blocked by cycloheximide. an inhibitor of protein synthesis.
• 4.4. The insulin stimulation of intracellular AIB accumulation is due to an increased influx and not to a reduced efflux of AIB.
• 5.5. Analysis of transport kinetics for AIB showed that insulin increased V_max but did not change K_m.
• 6.6. It is concluded that insulin stimulates uptake of certain neutral amino acids into kidney cortex cells in young animals.
• 7.7. The effect on renal amino acid transport appears to be mediated through increased synthesis of a membrane carrier.

     

The tolerance of lepidopterous larvae to an insect selective neurotoxin

The interaction of the fast-neurotoxic and insect selective polypeptide derived from scorpion venom (AaIT) with lepidopterous larvae tissues was studied through assays of toxicity, chromatography, binding and light microscopical autoradiography. The native and/or radioiodinated toxin was shown to:

• 1.(1) Induce a delayed, slow, progressive paralysis (within 24–48 h) of Spodoptera larvae by relatively high doses (paralytic unit = 2.4 μg/100 mg) corresponding to about only 10% of the total toxicity of the crude venom. Larvae of six species representing five families of Lepidoptera responded similarly to the toxin.
• 2.(2) Resist an in vitro incubation in the insect's hemolymph.
• 3.(3) Lose 80% of its toxicity in the insect's body within 24 h, accompanied by a progressive process of degradation and elimination by the excretory system.
• 4.(4) Specifically bind to a single class of non-interacting binding sites of high affinity and low capacity (0.2 pmol/mg protein, similar to tritiated saxitoxin) in an in vitro, homogenate derived, neuronal preparation.
• 5.(5) Specifically bind with high affinity to desheathed but otherwise intact nerves.
• 6.(6) Be devoid of accessibility to peripheral-terminal branches of Spodoptera motor nerves in situ—strongly contrasting those of the toxin susceptible Periplaneta nerves.

It may be thus concluded that the tolerance of the lepidopterous larvae to AaIT can be substantially attributed to pharmacokinetic aspects of toxin accessibility barriers and degradation processes.