Measurement of blood flow to osseous tissue in dogs using the radiolabelled microsphere method

• 1.1. The purpose of this study was to examine bone blood flow in various intra- and extra-oral sites.
• 2.2. The radiolabelled microsphere method was utilized to assess osseous blood flow in the following regions of 10 dogs: rib, long bone, and anterior and posterior regions of the maxilla and mandible.
• 3.3. Samples of cancellous and cortical bone were also obtained from each of these regions with the exception of the maxilla and the anterior mandible.
• 4.4. Mean blood flow ranged from 3.71 ±0.81 (SE) ml min.⁻¹ 100 g⁻¹ in the mandibular posterior cortical bone to 22.7±4.66ml min⁻¹ 100 g⁻¹ in the cancellous rib samples.
• 5.5. Blood flow to the cancellous tissue of the rib was significantly greater (P < 0.05 ) than the other tissues with the exception of maxillary posterior bone and cortical rib.
• 6.6. Results from this study indicate that blood flow to the maxillary posterior bone is relatively high, but blood flow in other intraoral osseous sites is significantly less than that of cancellous rib bone.

     

Combined effect of adenosine,alpha adrenergic and adenosine antagonists on serum insulin and insulin secretion from rat pancreatic islets

• 1.1. The effect of adenosine separately or in combination with alpha-1 adrenergic antagonist prazosin and alpha-2 adrenergic antagonist yohimbine as well as adenosine antagonists 8-phenyltheophylline and xanthine amine conjugate on glucose-induced insulin secretion from isolated rat pancreatic islets was studied.
• 2.2. Their in vivo effects on serum glucose and insulin levels were also investigated. Adenosine at 10 and 100 μM inhibited significantly, insulin secretion from the isolated islets whereas at 10 mM slightly increased the secretion of insulin.
• 3.3. Prazosin used at 100 μM inhibited insulin secretion. When it combined with adenosine (10 μM) it augmented the inhibitory effect of adenosine.
• 4.4. In vivo prazosin (21 mg/kg bodywt) caused a hyperglycaemia which was accompanied by hypoinsulinaemia.
• 5.5. Concurrent administration of this drug with adenosine neither affect the hyperglycaemic nor the hypoinsulinaemic effects of adenosine.
• 6.6. On the other hand, yohimbine (100 μM) has no effect neither separately nor in combination with adenosine (10 μM) in modulating the inhibitory effect of adenosine on insulin secretion.
• 7.7. When Yohimbine administered at 19.5 mg/kg body wt it did not alter serum glucose but it markedly increased the serum insulin level. Its combined administration with adenosine reduced the hyperglycaemic effect of adenosine with a remarkable increase in serum insulin.
• 8.8. Both adenosine-antagonists were ineffective in alteration of insulin secretion.
• 9.9. However, combination of 8-phenyltheophylline with adenosine (10 μM) totally blocked the inhibitory effect of adenosine on insulin secretion while xanthine amine conjugate failed to prevent this effect of adenosine.
• 10.10. These results indicate that the inhibitory effect of adenosine on insulin secretion is neither mediated via alpha-1 nor alpha-2 adrenoceptors. It might be via activation of specific adenosine receptors on rat islets which are sensitive to blockade by 8-phenyltheophylline.

     

Serum lipoproteins of sea bass (Dicentrarchus Labrax L.). Purification and partial characterization by density gradient ultracentrifugation and agarose column chromatography

• 1.1. Five classes of sea bass serum lipoproteins were purified by single vertical spin ultracentrifugation and agarose column chromatography
• 2.2. VLDL, beta migrating, are the larger and less dense lipoproteins.
• 3.3. LDL are the more heterogeneous in size, ranging from 11 × 10⁶ to 1 × 10⁶.
• 4.4. HDL represent the predominant class which, on the basis of density and electrophoresis migration, is differentiated in three subclasses.
• 5.5. VHDL float at a density > 1.22 mg/ml, which corresponds to the density of the other serum lipoproteins. This subclass, with an apparent molecular weight of 1.5 × 10⁵, resembles the albumin-like fatty acids binding proteins, shown in mammals and teleosts and absent in elasmobranchs.

     

Purification and properties of a β-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli

• 1.1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile “Caldocellum saccharolyticum strain Tp8T6.3.3.1. It was found to have an M_r of 42,000 and an isoelectric point of approx. 5.0.
• 2.2. The enzyme showed optimum activity at pH 5.0–7.7 and had an activation energy of 44 kJ mol⁻¹. It was stable at room temperature at pH 4.5–11.5 in the presence of 0.5 mg ml⁻¹ bovine serum albumin. The half-life of the enzyme at 75°C was 20 min at pH 6.0 in the presence of 0.5 mg ml⁻¹ bovine serum albumin.
• 3.3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The K_m for oat spelts xylan was 0.021% (w/v) at pH6.0.
• 4.4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside, but could not hydrolyse xylobiose.
• 5.5. It showed transferase activity on p-nitrophenyl β-D-xylopyranoside. Xylose did not inhibit the enzyme.

     

A comparative study of vertebrate eye lens crystallins using isoelectric focusing and densitometry

• 1.1. The crystallin proteins of numerous species belonging to different classes of vertebrates have been studied.
• 2.2. Species-specific crystallin patterns are revealed which unequivocally characterize the different species.
• 3.3. A marked variability in the number and percentage of alpha-, beta- and gamma-crystallins were found in the various species.
• 4.4. The gamma-crystallin family, with a meagre number of common bands, has proved to be most representative of the species. The beta-crystallins, with their greater number of common bands, have been best preserved throughout vertebrate evolution.
• 5.5. From the similarity coefficient matrix a dendrogram is drawn up, a visual phylogenetic summary of the interrelationships between the vertebrates considered.
• 6.6. In the Discussion, other aspects are considered, such as lens morphology, functionality, animal age, post-synthetic modifications and genetic factors.

     

Comparative examination of the soluble muscle proteins of two species of angler-fish (Lophiidae): Lophius piscatorius (L.) and Lophius budegassa (Spinola)

• 1.1. A comparative examination of sarcoplasmic proteins of the two nominal European species of angler-fish, Lophius piscatorius and L. budegassa was carried out using isoelectric focusing techniques.
• 2.2. Two protein bands differing in isoelectric point proved diagnostic for L. budegassa (pI 4.40 and pI 5.75) while a third characterized L. piscatorius (pI 4.65).
• 3.3. These species-specific protein profiles provide a method of species discrimination independent of morphological criteria.
• 4.4. Within-species heterogeneity of banding pattern suggested the presence of polymorphic gene loci of potential use in studies of population structure.

     

Increased muscle glucose uptake in response to chronic glyburide treatment is not related to changes in glucose transporter (GLUT4) protein

• 1.1. The mechanism of action of glyburide (a sulfonylurea) on muscle has been investigated by measuring glucose uptake and glucose transporter (GLUT4) protein levels after chronic glyburide treatment.
• 2.2. A dietary induced insulin resistant rat model (4 wk of high-fat, high-sucrose feeding) was given glyburide (2mg/kg/day) for 10 days and glucose uptake was measured in a perfused hindquarter preparation.
• 3.3. Protein levels of the GLUT4 glucose transporter were determined by Western analysis.
• 4.4. After 7 days of treatment, rats fed glyburide had lower blood glucose concentrations 2 hr (72 ± 5 vs 103 ± 12 mg/dl) and 24 hr (97 ± 7 vs 123 ± 7 mg/dl) after glyburide administration with no difference in serum insulin levels compared to vehicle treated animals.
• 5.5. Glucose uptake was approx doubled in basal state (0 insulin) in response to glyburide (2.8 + 0.4 vs 1.7 ± 0.2μ mol/g per hr).
• 6.6. Maximal insulin (100 nM) stimulated glucose uptake tended to be higher in the glyburide treated group, but did not reach statistical significance (8.0 ± 0.7 vs 7.0 ± 0.6 μmol/g per hr).
• 7.7. Western analysis revealed no significant effect of glyburide on the GLUT4 protein level in skeletal muscle.
• 8.8. These results suggest that glyburide alters glucose uptake through some mechanism other than alterations in the level of the GLUT4 glucose transporter protein.

     

Miniature gel filtration: a simple and inexpensive method for the measurement of protein- and non-protein-bound cortisol: application to guinea pig plasma

• 1.1. A method is described for the accurate and rapid measurement of protein- and non-protein-bound cortisol by miniature gel filtration in small volumes of plasma, e.g. of rodents.
• 2.2. Binding of cortisol by guinea pig plasma proteins is strongly reduced at elevated temperature (4°C: 102 ± 12ng/ml; 40°C: 5 ± 2 ng/ml).
• 3.3. Incubation of guinea pig plasma with 1–5000 ng cortisol resulted in a dose-dependent increase in cortisol bound to proteins (specific binding by corticosteroid binding globulin: 230 ± 12 ng/ml).
• 4.4. Administration of 20 IU (1–24)ACTH induced a significant increase of total protein-bound and non-protein-bound cortisol.
• 5.5. Values reported in this study agree well with those of previous investigations, in which bound and non-bound glucocorticosteroids were separated by gel filtration on large Sephadex® columns.

     

Solubilization,characterization and photoaffinity labeling of the mitochondrial dihydropyridine receptor from bovine adrenal medulla

• 1.1. The mitochondrial dihydropyridine receptor was solubilized with Chaps at a detergent/ protein ratio of 2.5, during 45 min at 4°C.
• 2.2. From the rate constants of association (8.10 ± 0.25 × 10⁴ M⁻¹ min⁻¹) and dissociation (0.022 ± 0.001 min⁻¹ a K_d of 275 nM was calculated, while from saturation experiments a K_d of 270 ± 30 nM and a density of receptors of 106 ± 9 pmol/mg protein was obtained.
• 3.4. The solubilized receptors are heat-resistant, sensitive to the trypsin and to the reduction of disulfide bonds.
• 4.5. In native membranes, a polypeptide of 50 kDa was specifically photolabelled with [³H]Azidopine.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Purification and characterization of lipoprotein lipase from the white adipose,skeletal muscle,cardiac muscle,mammary gland and lung tissues of the rat

• 1.1. Lipoprotein lipase (LPL) was isolated from five rat tissues: white adipose, skeletal muscle, cardiac muscle, mammary gland and lung.
• 2.2. Specific activity of the preparations varied from 75 U/mg for skeletal muscle and 720 U/mg for adipose.
• 3.3. The preparations were further analysed using SDS-PAGE and a single component identified. The mol. wt of 61,000 Da of this component was consistent for all five of the tissue sources.
• 4.4. Significant differences in the values of the isoelectric points of the enzyme species were revealed. The values varied from 7.23 (SEM 0.022) for cardiac and lung to 7.51 (SEM 0.037) for mammary.
• 5.5. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension and SDS-PAGE in the second revealed differences in the patterns of stained material derived from the five tissue sources.

     

Gluconeogenesis in hepatocytes determined with [2-13C] acetate and quantitative 13C NMR spectroscopy

• 1.1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using [2-¹³C]acetate and ¹³C magnetic resonance spectroscopy.
• 2.2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the ¹³C distribution in glucose where the overall ¹³C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean ¹³C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30.
• 3.3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20.
• 4.4. With this carbon exchange factor and the approximated ¹³C enrichment of acetyl-CoA the intramitochondrial ¹³C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated.
• 5.5. Since acetate was metabolized solely in liver cells the ¹³C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate.
• 6.6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively.
• 7.7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the ¹³C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%.
• 8.8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz [Katz J. (1985) Am. J. Physiol.248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis.
• 9.9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.

     

Effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail

• 1.1. The effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail were investigated.
• 2.2. The level of liver NAD was not affected by niacin deficiency whereas the level of pectoral muscle NAD was markedly reduced.
• 3.3. In all dietary treatments the liver had the highest turnover rates of proteins, heart and brain had intermediate rates, and pectoral muscle had the lowest rates.
• 4.4. Relative turnover rates of proteins in all tissues (particularly pectoral muscle) of the niacin deficient group were significantly higher than those of pair-fed control group, although there were no significant differences in turnover rate between pair-fed control and control groups.
• 5.5. The high turnover rate of proteins in niacin deficiency was primarily attributed to enhanced degradation rate of proteins rather than enhanced synthesis rate of proteins.
• 6.6. Optical density scanning (or densitometric) of water-soluble pectoral muscle proteins separated by isoelectric focusing revealed several additional minor protein bands between major protein bands in the niacin deficient group which were more pronounced in the acidic region of the gel.
• 7.7. These results suggest that proteins with a low pI value in pectoral muscle of the niacin deficient animal are highly sensitive to protein degradation.

     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

A Robust and Versatile Automated Glycoanalytical Technology for Serum Antibodies and Acute Phase Proteins: Ovarian Cancer Case Study

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Highlights

• •Quantitative high-throughput glycoanalytical technology as a diagnostic tool for ovarian cancer detection.
• •Multiplexed approach harnessing N-glycan data for six glycoproteins from a single biological sample.
• •Detailed characterization of human serum N-glycans from antibodies IgG, IgM and IgA and acute phase proteins transferrin, haptoglobin and alpha-1-antitrypsin.
• •Structural differences in antibody and acute phase protein glycosylation for mechanistic insights.

     

The induction of α1-acid glycoprotein by methylmercury

• 1.1. The incorporation of [¹⁴C]leucine into protein was measured in liver preparations and blood of rats following the s.c. administration of methylmercury hydroxide (24 mg/kg body wt) or turpentine (5.0 ml/kg body wt).
• 2.2. The translatability of the RNA obtained from polysomes in an mRNA-dependent reticulocyte lysate was elevated significantly in the preparations derived from the treated rats compared to control rats.
• 3.3. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of α₁-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.

     

Purification and characterization of a novel chymotrypsin inhibitor controlled by the chymotrypsin inhibitor A (Ict-A) gene from the larval hemolymph of the silkworm,Bombyx mori

• 1.1. On polyacrylamide gel electrophoresis, chymotrypsin inhibitors in the larval hemolymph of the silkworm were found as 15 electrophoretically distinct bands.
• 2.2. One of them, CI-13 migrating fastest toward the anode, was purified from the hemolymph.
• 3.3. The inhibitor was a monomeric protein with a molecular mass of 14,000 and showed stability for both heat and a wide range of pH. The isoelectric point was pH 4.1.
• 4.4. CI-13 was able to inhibit chymotrypsin completely and trypsin slightly, but was ineffective against other proteinases such as papain, ficin, carboxypeptidase A, V8 proteinase, serratia peptidase, cocoonase and proteinases derived from gut juice of the silkworm.

     

Sodium transport in red blood cells of lamprey LAmpetra fluviatilis

• 1.1. Unidirectional Na⁺ influx in lamprey red blood cells was determined using ²²Na as a tracer.
• 2.2. Total Na⁺ uptake and amiloride-inhibitable Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration (Na_e).
• 3.3. At 141 mM Na_e, the average value of net Na⁺ influx was 13 ± 1.1 and the amiloride-sensitive Na⁺ influx was 5.3±1.1 mmol/l cells per hr (±SE).
• 4.4. The amiloride-sensitive component of Na⁺ influx was significantly activated by 10⁻⁵ M isoproterenol, by 2 × 10⁻⁵ M DNP, and by cell shrinkage.
• 5.5. Furosemide (1 mM) had no effect on the Na⁺ transport in red cells.
• 6.6. The residual amiloride-insensitive component of Na⁺ transport was a linear function of Na_e in the range of 5–141 mM. This transport seems to be accounted for by simple diffusion.

     

Serum ions concentration and atpase activity in gills,kidney and oesophagus of european sea bass (Dicentrarchus labrax,pisces, perciformes) during acclimation trials to fresh water

• 1.1. Kidney, oesophagus and gill Na⁺-K⁺ ATPase activity and serum Na⁺, K⁺ and Cl⁻ concentrations are evaluated in European sea bass during experimental acclimation to fresh water.
• 2.2. Kidney and oesophagus ATPase increase in low salinity and reach a maximum in fresh water.
• 3.3. Gill ATPase decreases during the acclimation trials and rises again to normal values after a 3-week stay in fresh water.
• 4.4. Na⁺ and K⁺ serum concentrations decrease during the trials and increase back after a 3-week stay in fresh water.
• 5.5. The correlations between enzymatic activities, serum ion concentrations, morphological changes and environmental salinity are discussed.

     

Age-dependent proteins in eyes of annual killifishes Pterolebias longipinnis detected by 2-dimensional electrophoresis

• 1.1. Eye proteins of Pterolebias longipinnis have been analyzed by 2-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis during aging from adolescence until normal death.
• 2.2. The protein pattern on the gels changed gradually with progressing age.
• 3.3. In senescent eyes, three protein spots appeared for a time and 36 disappeared from the pattern.
• 4.4. The isoelectric points of the proteins in the presence of urea and the molecular weights in an unreducing buffer are presented.