Variation in plasma constituents during the natural vitellogenic cycle of tuatara,Sphenodon Punctatus

• 1.1. The vitellogenic cycle of female tuatara was investigated by monitoring plasma levels of vitellogenin, calcium, total protein, inorganic phosphate (P₁) and cholesterol.
• 2.2. Vitellogenin was not detected in females in the non-reproductive condition, but was found perenially in plasma of reproducing females during vitellogenesis, which normally lasts about 3 years out of the 4 year ovarian cycle.
• 3.3. No large year-to-year variations were found in the plasma constituents measured and there was no correlation between the oestradiol peak at mating and plasma levels of vitellogenin.
• 4.4. The results provide further evidence that tuatara have an extraordinary prolonged and gradual vitellogenic cycle spanning several years for a single clutch of eggs. This type of reproductive cycle is unique among reptiles.

     

Sulphhydryl protease inhibitors from pineapple plant stem

• 1.1. Pineapple stem extract, consisting of a mixture of the protease bromelain and sulphhydryl protease inhibitors, was fractionated by gel permeation chromatography.
• 2.2. Inhibitor-containing fractions were further resolved by ion exchange chromatography on DEAE-cellulose, giving 12 chromatographically distinct inhibitory fractions.
• 3.3. These 12 inhibitory fractions all show an inhibition specificity towards bromelain.
• 4.4. Reduction, S-carboxymethylation and refractionation of each of these inhibitory fractions gave, for each fraction, two separated peptides of ca 13 and 40 amino acids in length, respectively.
• 5.5. The amino acid compositions and the N-terminal sequences of these peptides show the inhibitors to be a closely homologous set. Both the constituent peptides of each fraction are microheterogeneous. Each DEAE-cellulose chromatogram peak contains a co-eluting set of iso-inhibitors.
• 6.6. Structural microvariations within these isoinhibitors have a minor influence on inhibitor activity towards bromelain.

     

Isolation and characterization of new minor proteins (Ovoglycocomponents) from chicken and quail egg whites

• 1.1. New minor proteins were isolated from chicken and quail egg whites and were named the ovoglycocomponent.
• 2.2. They migrated on polyacrylamide-gel electrophoresis as a single band without any contaminations.
• 3.3. They contain a considerable amount of carbohydrates, of which hexosamine was much higher in the chicken than in the quail.
• 4.4. The molecular weights of the chicken and quail ovoglycocomponents estimated from gel filtration were approx 56,000 and 49,000, respectively.
• 5.5. The isoelectric points were measured as 5.1 for the chicken and 5.4 for the quail.

     

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.

     

The isolation of skeletal muscle plasma membranes from rainbow trout (Salmo gairdneri)

• 1.1. Plasma membranes were isolated from caudal flank skeletal musculature of rainbow trout by discontinuous sucrose gradient centrifugation.
• 2.2. Na⁺−K⁺-ATPase was enriched 8-fold and 5′-nucleotidase activities 4-fold in a fraction isolated at the 8–25% sucrose interface.
• 3.3. A cholesterol: phospholipid ratio of 0.37 in the plasma membrane fraction was 85% greater than that observed in adjacent subcellular fractions.
• 4.4. Electron microscopy provided morphological confirmation of enrichment and integrity of skeletal muscle plasma membranes at the 8–25% sucrose interface.

     

Sex-specific androgen binding in spotted hyaena (Crocuta crocuta) plasma

• 1.1. A charcoal adsorption assay demonstrated a large variance in androgen binding ability in female spotted hyaenas.
• 2.2. A positive correlation between plasma androgen binding ability and ovarian steroid concentrations was demonstrated in adult females.
• 3.3. The strong plasma binding affinity for testosterone and dihydrotestosterone (DHT) (nM) together with the lack of cortisol and weaker oestradiol-17β binding suggests that a specific androgen binding substance, possibly a protein, is present in adult females of this species.
• 4.4. The lack of high affinity binding in male spotted hyaenas is unusual and deserves further investigation.
• 5.5. Some androgen binding in all, including males and immature animals suggests that albumin may bind some plasma androgens in this species.

     

Synthesis and maturation of the major yolk protein by the ovaries of the firebrat,Thermobia domestica (Insecta,thysanura)

• 1.1. Ovaries of Therobia domestica, dissected from inseminated females and incubated with tritiated amino acids, synthesize labeled proteins, the major fraction of which is indistinguishable from the major vitellogenin secreted by the fat body, when considering the electrophoretic mobility, the polypeptide composition and the immunoreactivity.
• 2.2. Peptide mapping, using two different proteases, shows a striking structural similarity between the proteins of both origins and reveals interrelationships between their subunits.
• 3.3. The ovary synthesizes the 210–212 kD precursors of the major vitellogenin, as does the fat body, and processes them intensively into smaller subunits (176–182, 57 and 46 kD). The follicle cells are tentatively nominated for both roles.
• 4.4. The quantitative contribution of the two ovaries to the vitellogenin pool was found to be much higher than that of the fat body.

     

Immunocytochemical localisation of vitellogenic and non-vitellogenic yolk proteins in the ovary of leptinotarsa decemlineata

• 1.1. Two vitellogenins and chromoprotein 2 are selectively accumulated by the oocyte and cannot be detected either in follicle cells or in the germarium.
• 2.2. At the start of their accumulation in terminal oocytes they are asymmetrically distributed.
• 3.3. Endocytosis of vitellogenin 1 starts somewhat later than the uptake of vitellogenin 2 and chromoprotein 2.
• 4.4. In follicle cells of young follicles, a protein (DLP), immunologically related to diapause protein 1, is highly concentrated.
• 5.5. During vitellogenesis DLP is sequestered by the oocytes.
• 6.6. The protein rich globules in terminal oocytes contain the vitellins as well as chromoprotein 2 and DLP.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

     

Plasma lipoprotein profiles and arylesterase activities in two inbred strains of rabbits with high or low response of plasma cholesterol to dietary cholesterol

• 1.1. Cholesterol feeding for 4 weeks of female and male rabbits of two inbred strains increased plasma cholesterol concentrations by about 11 and 48 mmole/I in the hypo- and hyperresponsive strain, respectively.
• 2.2. On the low-cholesterol pre-experimental diet, the hyporesponsive animals had significantly higher plasma HDL (high density protein) cholesterol levels than hyperresponders.
• 3.3. In both strains, cholesterol feeding caused elevations of cholesterol in all lipoprotein classes, the difference between the hypo- and hyperresponsive strains in essence only being observed in the VLDL (very low density lipoprotein) fraction.
• 4.4. Basal plasma total arylesterase activity was significantly higher in the hypo- than in the hyperresponsive rabbits.
• 5.5. Dietary cholesterol caused an increase in plasma esterase activity in both strains.
• 6.6. We suggest that in rabbits a low plasma arylesterase activity and a low concentration of HDL cholesterol are associated with an increased sensitivity to dietary cholesterol.

     

Subcellular distribution of metals within the kidney of the bivalve Mercenaria mercenaria (L.)

• 1.1. The subcellular distribution of nine transition metals (plus four additional elements) was measured in the kidney tissue of the quahog, Mercenaria mercenaria.
• 2.2. Elemental analyses of the subcellular fractions indicated three main patterns of metal distribution within kidney cells.
• 3.3. Barium, iron, manganese and lead were associated primarily with kidney granules.
• 4.4. Cadmium, copper, potassium and magnesium were found mainly in the cytosolic fraction.
• 5.5. Calcium, phosphorus and zinc were found in all isolated fractions, probably reflecting the important roles that these elements play in bivalve metabolism.
• 6.6. The organelle composition of the isolated subcellular fractions was determined using marker enzyme assays and microscopic techniques.

     

Differentiation between carrier-bound forms of non-suppressible insulin-like activity (NSILA) in serum

• 1.1. Gel-permeation chromatography of serum on Sephacryl S-300 at pH 7.4 has shown that NSILA was detected over a range of MW 50,000–400,000 with a peak at about MW 200,000.
• 2.2. When fractions from the above chromatography were rechromatographed on Sephadex G-75 at pH 2.4 major amounts of acid-stable NSILA were found in a fraction of MW 200,000–600,000 (77% of the fraction NSILA or 28% of total serum NSILA).
• 3.3. Further evidence was obtained for the presence of an active acid-dissociable complex in serum. This was present in both the MW 100,000–200,000 and 35,000–100,000 fractions and corresponded to 37% of total serum NSILA.
• 4.4. Con-A Sepharose affinity chromatography of the serum fractions from Sephacryl S-300 chromatography, followed by Sephadex G-75 chromatography under acid conditions, showed that the acid-stable complex was consistently found in weakly bound materials. The active acid-dissociable complex was found in the bound fractions, especially in the Sephacryl S-300 pool of MW 35,000–100,000.
• 5.5. Low MW NSILA (<15,000) was also released on acid treatment from an otherwise inactive high MW complex(es) of MW 35,000–600,000. This complex was not bound by Con-A Sepharose.

     

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

A survey of the non-esterified fatty acids and binding proteins in the plasmas of selected animals

• 1.1. The levels of non-esterified fatty acids (NEFA) in the plasma of a variety of animals have been estimated.
• 2.2. Only one of seven elasmobranchs contained detectable levels of NEFA.
• 3.3. The two crustaceans examined contained very low levels.
• 4.4. All the other animals contained circulating levels of a variety of NEFA ranging from 14 to 24 carbon atoms.
• 5.5. The elasmobranchs are unique in that they also do not possess proteins in the serum which bind fatty acids.

     

Electrophoretic patterns of yolk proteins throughout ovarian development and their relationship to vitellogenin in the rainbow trout,Oncorhynchus mykiss

• 1.1. Electrophoretic patterns of yolk proteins were investigated throughout ovarian development and their relationship to vitellogenin determined in a pulse-chase experiment with ³H-vitellogenin.
• 2.2. Using a radioimmunoassay for vitellogenin, vitellogenin/yolk protein products of vitellogenin were detected in follicles throughout ovarian development and in ovulated eggs.
• 3.3. The majority of yolk proteins in follicles measuring less than 1.0 mm in diameter appeared to be derived from sources other than vitellogenin. In contrast, in the larger follicles all of the major yolk proteins detected were derived from vitellogenin.
• 4.4. Pulse-chase with ³H-vitellogenin revealed that all of the major yolk proteins in 3.0 mm follicles were derived from vitellogenin. The major peptides eluted with molecular masses of 110 and 30 kDa under non-reducing conditions (these are very likely to represent lipovitellin 1 and lipovitellin 2), and 88, 22, 16, and 12 kDa under reducing conditions.
• 5.5. There were no apparent differences in the major yolk proteins in ovulated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis of these proteins had occurred during maturation and/or ovulation.

     

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.

     

Comparative study of ovalbumins from various avian species by Con A/Sepharose chromatography

• 1.1. Ovalbumin samples from 7 chicken subspecies and other avian species were subfractionated on Con A/Sepharose column.
• 2.2. The ovalbumins from chicken subspecies were separated into 4 fractions, OA, OB, OC and OD without exceptions, although the fractional ratio (OA:OB:OC:OD) was varied from one sample to another. Similarly, turkey ovalbumin also could be separated into the 4 fractions.
• 3.3. Quail ovalbumin was tightly bound to Con A/Sepharose column and effectively eluted as a new characteristic peak (OQ) behind OD from the column at room temperature, while goose ovalbumin was totally eluted as unadsorbed fraction from the column.

     

Rat brain aryl acylamidase: Further characterization of multiple forms

• 1.1. Two fractions of aryl acylamidase (EC 3.5.1.13) were further separated from rat brain extracts at pH 7.5 by ammonium sulfate precipitation and Bio-Gel chromatography.
• 2.2. 1,2,3,4-Tetrahydro-β-carboline competitively inhibited (67%) fraction-1 but slightly inhibited (13%) fraction-2. Tetrahydroharman, 6-hydroxy-tetrahydroharman and harminic acid slightly inhibited both fractions. Harmalol inhibited fraction-1 but enhanced fraction-2. 6-Methoxy-harman, 6-methoxy-harmalan and harmaline enhanced both fractions.
• 3.3. Pargyline did not affect either fraction. Methiothepin, cyproheptadine and chlorimipramine inhibited fraction-1 but stimulated fraction-2.
• 4.4. Neostigmine moderately (30%) inhibited AAA-2 but did not have any significant effect on AAA-1.
• 5.5. These results indicate that the β-carboline compounds might play a role in regulating activity of AAA-1 and 2 in brain.
• 6.6. Both fractions might be related to serotonergic neurons but only AAA-2 might be associated with acetylcholinesterase.

     

Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

• 1.1. Four GTP-binding proteins (23–27 kDa) were identified in membranes from PC12 cells by [α³²P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels.
• 2.2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K⁺-depolarization or even after addition of C²⁺to digitonin-permeabilized cells.
• 3.3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca²⁺.
• 4.4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity.
• 5.5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane.
• 6.6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

     

Phenoloxidase activity of acridid grasshoppers from the subfamilies melanoplinae and oedipodinae

• 1.1. Phenoloxidase activity and wound melanization was studied in five species of grasshoppers representing the subfamilies Melanoplinae and Oedipodinae.
• 2.2. Most of the phenoloxidase activity was detected in the plasma fraction of grasshopper whole-body homogenates and supernatant fractions of the hemolymph. The species representing the Oedipodinae had 20–50% higher percentage of the total phenoloxidase activity associated with particulate matter from a whole-body homogenate when compared to the Melanoplinae.
• 3.3. Phenoloxidase activity could not be detected in sclerotized cuticle of adult grasshoppers.
• 4.4. The phenoloxidase existed as a zymogen which could be activated by chymotrypsin and inhibited by KCN and NaCN while EDTA showed no effect. It had optimum activity at 37°C and pH 7.3.
• 5.5. These findings are discussed in relation to wound repair and immune responses to infection in grasshopper species.