Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline

• 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-P_i basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
• 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
• 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
• 4.4. The method could also be applied to purify PLC produced in a low-P_i complex medium. The resultant preparation was not homogeneous.
• 5.5. The molecular weight for both PLC preparations was about 70 kDa.
• 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K_M of 25 mM for p-nitrophenylphosphorylcholine.
• 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
• 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP_l-BSM plus choline but not the enzyme from the LP_l-CM.

     

In vitro Ca deposition by rat matrix vesicles: Is the membrane association of alkaline phosphatase essential for matrix vesicle-mediated calcium deposition?

• 1.1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media.
• 2.2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment.
• 3.3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment.
• 4.4. The specific activity of the released ALP was at least 5-fold higher than the residual activity.
• 5.5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or βGP-dependent calcium deposition.
• 6.6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.

     

Effects of short chain fatty acid,sodium butyrate,on osteoblastic cells and osteoclastic cells

• 1.1. Sodium butyrate increased alkaline phosphatase (ALP) activity of cloned osteoblastic cell line MC3T3-El by the stimulation of de novo enzyme synthesis.
• 2.2. Sodium butyrate did not affect mature osteoblastic cells but affected preosteoblastic cells.
• 3.3. Sodium butyrate decreased tartrate resistant acid phosphatase (TRACP)-positive multinucleated cells (MNC) formation from bone marrow cells. This related to the cytotoxicity of sodium butyrate on bone marrow cells.

     

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: Hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of tris

• 1.1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH.
• 2.2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint.
• 3.3. Individual constants of rate of a simple transphosphorylation mechanism have been determined.
• 4.4. At high Tris concentrations (> 1.0 M) a slight competitive inhibition has been observed.
• 5.5. Inhibition in NH₄⁺-NH₃Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH₂ group of Tris is responsible for inhibition.
• 6.6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.

     

Differential vitamin and choline requirements of symbiotic and aposymbiotic s. oryzae (coleoptera: curculionidae)

• 1.1. Development times, fertilities and weights of symbiotic and aposymbiotic strains of the rice weevil Sitophilus oryzae in response to single omissions of seven vitamins and choline were studied.
• 2.2. No strict requirement for choline could be demonstrated: development times and weights were comparable to diets with or without choline, but the presence of choline increased fertility of the symbiotic strain on vitamin-deficient diets.
• 3.3. The 0.02% concentration used in these experiments may be too high since it resulted in a lower fertility of the aposymbiotic strain.
• 4.4. The results indicated that niacin could be synthesized by the weevils.
• 5.5. Thiamine, folic acid and pyridoxine were found to be required in the diet of both strains but the latter two may be supplied in part by symbiotes since a first symbiotic generation was obtained.
• 6.6. Pantothenic acid, biotin and riboflavin were only required by aposymbiotic weevils: their omission from the diet did not modify the development of symbiotic insects.
• 7.7. It is therefore suggested that these three vitamins are supplied by symbiotes.

     

Acid and alkaline phosphomonoesterase activities in snake venoms

• 1.1. Acid and alkaline phosphatase activities of eight different snake venoms were determined quantitatively by using synthetic substrates, o-carboxyphenylphosphate and p-nitrophenylphosphate respectively.
• 2.2. It was found that most of Elapidae venoms investigated had both acid and alkaline phosphatase activities.
• 3.3. Three Crotalidae venoms investigated did not show any alkaline phosphatase activity.
• 4.4. The strength of venom acid phosphatase activity is as follows: Agkistroden acutus > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.
• 5.5. The strength of venom alkaline phosphatase activity by using p-nitrophenylphosphate is in the order of Naja hannah > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.When o-carboxyphenylphosphate was used as a substrate, the order of enzyme activity is Naja hannah > Naja haje > Naja naja samarensis > Naja melanoleuca > Naja naja atra.
• 6.6. Acid phosphatase activity of all the Elapidae venoms was inhibited completely by fluoride. The alkaline phosphatase activity of Elapidae venoms was not inhibited by fluoride either using p-nitrophenylphosphate or o-carboxyphenylphosphate.
• 7.7. The acid phosphatase of all the Elapidae venoms was not inhibited by zinc ion. However, most of the venom alkaline phosphatases were inhibited by zinc ion.
• 8.8. Ethylenediaminetetraacetic acid (EDTA) had inhibitory action on venom phosphatase activity. However, tris-(hydroxymethyl)-aminoethane had a counter effect on the inhibitory action of EDTA.
• 9.9. Optimum pH studies of the snake venom phosphatases showed that the acid phosphatases of the snake venoms had their highest activity in the range of pH 4–5. The alkaline phosphatases of the snake venoms had their optimum pH at 9.
• 10.10. Comparable experiments were also conducted by using chicken intestine alkaline phosphatase and wheat germ acid phosphatase.

     

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.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Electrical transport characteristics of Aplysia californica intestinal epithelium

• 1.1. Replacing chloride (Cl⁻) with sulfate (SO₄²⁻) in the bathing medium drastically reduced the mucosal membrane potential difference (ψ_m).
• 2.2. The voltage divider ratio was significantly greater than one.
• 3.3. Mucosal ^d-glucose decreased the input resistance of the intestinal epithelium.
• 4.4. Addition of furosemide to the mucosal bathing medium inhibited transepithelial potential difference and short-circuit current.
• 5.5. Addition of SITS to the mucosal bathing medium partially inhibited transepithelial potential difference and short-circuit current.
• 6.6. Diffusion potentials in the intestinal epithelium were symmetrical.

     

Alkaline phosphatase from Basidiobolus haptosporosus: Comparison with alkaline phosphatases from rainbow lizard and man

• 1.1. DEAE-cellulose chromatography of mycelial alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from Basidiobolus haptosporosus, produced three iso-enzymes “A”, “B” and “C”.
• 2.2. Fraction “A” was further characterized and showed maximum activity at pH 10 in 0.1 M sodium carbonate-bicarbonate buffer.
• 3.3. The enzyme was stimulated by Mg²⁺, Co²⁺ and Mn²⁺ and inactivated by Zn²⁺, Cu²⁺, EDTA, citrate and tartrate.
• 4.4. Phosphate ions inhibited it competitively, phenylalanine uncompetitively and urea noncompetitively.
• 5.5. It was heat stable for 60 min at 37°C but labile above 55°C.
• 6.6. Its K_m with p-nitrophenylphosphate was 0.5 mM; its estimated molecular weight was 160,000.
• 7.7. The results are compared with the properties of alkaline phosphatases from the rainbow lizard and man and discussed in terms of a triadic association between the fungus, the lizard and man.

     

Isolation and characterization of the major phospholipase a2 from the venom of trimeresurus purpureomaculatus (shore pit viper)

• 1.1. The major phospholipase A₂ (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity.
• 2.2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis.
• 3.3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme.
• 4.4. The phospholipase A₂ exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.

     

The effect of temperature on the acid-base status of the blood of the hatching quail (Coturnix c. japonica)

• 1.1. The ambient temperature of embryos of pipped eggs was reduced from 38 to 28°C for a period of 45 min.
• 2.2. The blood P_CO2 was lower and the blood more alkaline at 28°C than at 38°C.
• 3.3. At 28°C plasma [HCO₃⁻] ] was lower than predicted from the blood buffer line determined in vitro.
• 4.4. The plasma concentrations of strong ions and lactate were the same at both temperatures.
• 5.5. After the ambient temperature had been returned to 38°C for a period of 45 min, blood pH was more acidic than before cooling, but there was no difference in blood P_CO2.
• 6.6. The plasma [HCO₃⁻] was the same as that at 28°C and plasma [K⁺] was higher than before cooling.
• 7.7. The results arc discussed in relation to the factors affecting blood pH in embryos at this stage of development.

     

The effect of cortisol on stimulation of enzymatic activity and absorption of amino acids in the small intestine of adult hamsters

• 1.1. The s.c. administration of cortisol to hamsters (50 mg/kg body wt/day for 4 days) produces a significant increase in maltase sucrase, alkaline phosphatase and leucineaminopeptidase activity in intestinal mucosa.
• 2.2. Lactase activity is unaffected by cortisol.
• 3.3. Gamma-glutamyltranspeptidase activity increases slightly in females but remains unchanged in males.
• 4.4. Cortisol causes increase in proline and glycine absorption without changing the absorption of lysine.

     

Lactoferrin binding to human platelets

• 1.1. Platelets bind specifically lactoferrin.
• 2.2. The lactoferrin binding to the platelets depends on the concentration of labelled lactoferrin, the number of platelets, the time of incubation and pH.
• 3.3. The binding was characterized by two types of binding site: one with high affinity and low capacity, and another with low affinity and high capacity (respectively k_{aff 1} = 13.6 × 10⁹1/mol and about 40 binding sites, and K_{aff 2} = 1.23 × 10⁹1/mol and about 135 binding sites per platelet).
• 4.4. Both human transferrin and bovine lactoferrin compete with human lactoferrin for the receptors.
• 5.5. The presence of lactoferrin receptors on the platelet membrane surface is connected most probably with the effect(s) on the cell function(s) of these cells.

     

Human sciatic nerve phospholipid profiles from non-diabetes mellitus,non-insulin-dependent diabetes mellitus and insulin-dependent diabetes mellitus individuals. A 31P NMR spectroscopy study

• 1.1. Human sciatic nerve phospholipids obtained from non-diabetes mellitus (NDM), non-insulin-dependent diabetes mellitus (NIDDM), and insulin-dependent diabetes mellitus (IDDM) patients, after lower extremity amputation, were studied by ³¹P NMR spectrometry.
• 2.2. Nine phospholipids resonances in NDM and NIDDM groups were identified as followed: Ethanolamine plasmalogen (Epias, Chemical shift = 0.07δ); phosphatidylethanolamine (PE, 0.03δ); phosphatidylserine (PS, −0.05δ); sphingomyelin (SM, −0.09δ); lysophosphatidylcholine (LPC, −0.28δ); phosphatidylinositol (PI, −0.30δ); alkylacylphosphorylcholine (A1.PC, -0.78δ); phosphatidylcholine (PC −0.84δ), and an unknown resonance (U, 0.13δ).
• 3.3. In the IDDM group a resonance of lysophosphatidylinositol (LPI, 0.01δ) was detected in addition to the nine phospholipids listed above.
• 4.4. IDDM showed that PI and Al.PC were elevated and U was lower when compared with NDM; also, Eplas was lower when compared with NIDDM. PC was elevated and PS was lower when compared with both NDM and NIDDM.
• 5.5. Indices calculated from this data, showed that the choline ratio and choline/ ethanolamine ratio were elevated; while ethanolamine ratio, and myelin ratio were lower in IDDM group, when compared with both NDM and NIDDM groups.
• 6.6. Inactivation of the cholineacethyltransferase enzyme (ChAT) and enhancement of the phospholipidmethyltransferase enzyme (PLMT), secondary to an insulin deficiency, are proposed as an interpretation of these findings.

     

Breast,hips, and buttocks revisited: Honest fatness for honest fitness

This paper comments on: Low, B. S., Alexander, R. D., and Noonan, K.M. Human hips, breast, and buttocks: Is fat deceptive? Ethology and Sociobiology 8: 249-247, 1987. In it I argue that:

• 1.1. Sexual selection has probably not been the most important selection pressure on
• 2.female human body shape.
• 3.2. Male humans in different cultures find different aspects of the female body attractive
• 4.and therefore are unlikely to have exerted consistent directional sexual selection on
• 5.the female body.
• 6.3. Breast size is not correlated with lactation success.
• 7.4. Visible hip width is not correlated with parturition success.
• 8.5. Women would lower their fitness if they tried to deceive men about their internal
• 9.pelvic dimensions.
• 10.6. There are many alternative hypothesis to explain the existence of fat onwomen's
• 11.breast, hips, and buttocks.

     

Respiration in the eastern water dragon,Physignathus lesueurii (agamidae)

• 1.1. Some aspects of the gas exchange system of a diving lizard, Physignathus lesuewii were studied.
• 2.2. Breathing patterns were analysed.
• 3.3. Breathing rate increases logarithmically with temperature and Q₁₀ = 1.8. LogBR = −0.237 + 0.0256 T.
• 4.4. Gas tensions in lung air and arterial and venous blood were measured. Arterial pH declines with increasing temperature.
• 5.5. Temperature has a marked effect on oxygen affinity of the blood (ΔH = −10.1 kcal mol⁻). A Bohr effect was also noted.
• 6.6. CO₂ equilibrium curves were drawn.
• 7.7. The results are considered with a view to anticipating the efficiency of the gas exchange system of this species under conditions of variable temperature and during diving.

     

Characterization of chicken intestinal brush border membrane Ns/H exchange

• 1.1. Na/H exchange is the major pathway for Na uptake in brush border membrane vesicles from chicken small intestine. Hanes-Woolf analysis demonstrated that Na and H competed at the same extravesicular site. The K_Na for Na⁺ at extravesicular pH 6.6 is 35 mM and at pH 7.4, 12 mM.
• 2.2. Similar to mammalian intestinal cells, the Na/H exchanger does not appear to have an internal proton modifier site. Varying intravesicular pH from 6.1 to 7.8 stimulates uptake, but a sigmoidal relationship is not observed.
• 3.3. The ability of several amiloride analogs to inhibit the exchanger was tested and the inhibitory profile was similar, but not identical to Na/H exchangers in mammalian tissues. The potency series (from most to least potent) is hexamethylamiloride ≈ ethylisopropylamiloride > methylisobutylamiloride > dimethyl-amiloride > amiloride.