Antioxidant activity of the simulated gastro-intestinal digestion hydrolysate of two edible Nigerian marine molluscs: Tympanatonus fuscatus var radula (L.) and Pachymelania aurita (M.)

The multifunctional nature of antioxidant peptides makes them more attractive candidates as dietary ingredients in health maintenance. Therefore, food protein-derived antioxidant peptides are continuously investigated. This study investigated the in vitro antioxidant properties of hydrolysate and ultrafiltered peptide fractions of Pachymelania aurita and Tympanatonus fuscatus var radula-two commonly consumed marine molluscs known as periwinkles in southern Nigeria. Simulated gastrointestinal digestion (SGID) of soluble proteins of T. fuscatus and P. aurita was carried out using pepsin, trypsin and chymotrypsin, and the SGID hydrolysates were fractionated using a 3 kDa membrane filter. The hydrolysates and their fractions were investigated for anti-lipid peroxidation, hydroxyl radical scavenging activity (HRSA), ferric reducing antioxidant property (FRAP) and metal chelation activity, and they demonstrated clear antioxidant properties in all the assay models used. Low molecular weight fractions of the hydrolysates demonstrated more potent antioxidant activity than higher molecular weight fractions. This is profound in the metal chelation assay, where low molecular weight peptide fractions, T ≤ 3 kDa and P ≤ 3 kDa (IC₅₀ values of 8.10 ± 0.011 and 5.56 ± 0.50 µg/ml respectively) had activity that is similar to that of EDTA (11.84 ± 0.89 µg/ml). Similar activity effects were observed in other assays where there was about 3-fold higher activity in low molecular weight fractions. These results demonstrate the presence of antioxidant peptide(s) in the protein hydrolysates of the periwinkles.     

Some features of mitochondria and fluffy layer in regenerating rat liver

1. Mitochondria and fluffy layer were prepared from control and regenerating rat liver. Differential and density-gradient centrifugation were used to fractionate the preparations, which were examined for protein content, density and the activity of cytochrome c oxidase, succinate dehydrogenase, NAD–isocitrate dehydrogenase and NADP–isocitrate dehydrogenase. 2. During regeneration the mitochondrial protein content of the liver fell by 18% from the control value of 18·4mg. of protein/g. of liver (wet wt.) and by 3 weeks had risen to 130% of the control value. It then declined slowly. 3. The fluffy-layer protein content (4·7mg./g. of liver) varied inversely as the mitochondrial content and increased by 70% in the early stages (10 days) of liver regeneration. The results suggest that fluffy layer may partially represent both partly formed and broken-down mitochondria. 4. NAD– and NADP–isocitrate dehydrogenases differed in their behaviour during liver regeneration. 5. The succinate-dehydrogenase and NADP–isocitrate-dehydrogenase activity of fluffy layer was high and rose during the early stages of liver regeneration (1 week). Succinate dehydrogenase and cytochrome c oxidase were concentrated in the lighter fluffy-layer particles 10 days to 3 weeks after partial hepatectomy. The significance of this with respect to mitochondrial formation is discussed. 6. Mitochondrial fractions possessed a certain degree of heterogeneity in enzymic activity when separated according to size and density. The mean density of heavy mitochondria was 1·198, light mitochondria 1·193. Fluffy layer was nearly homogeneous in control liver, but during regeneration considerable heterogeneity became evident. The significance of the heterogeneity is discussed.     

Impaired aerobic work capacity in insulin dependent diabetics with increased urinary albumin excretion

To assess whether decreased aerobic work capacity was associated with albuminuria in insulin dependent diabetics aerobic capacity was measured in three groups of 10 patients matched for age, sex, duration of diabetes, and degree of physical activity. Group 1 comprised 10 patients with normal urinary albumin excretion (<30 mg/24 h), group 2 comprised 10 with incipient diabetic nephropathy (urinary albumin excretion 30-300 mg/24 h, and group 3 comprised 10 with clinical diabetic nephropathy (urinary albumin excretion >300 mg/24 h). Ten non-diabetic subjects matched for sex, age, and physical activity served as controls. Oxygen uptake was similar in the four groups at rest and during a 75 W workload. Maximal oxygen uptake was also similar in the control subjects and group 1 (median 41·7, (range 29·1-53·0) ml/kg/min v 38·5 (26·6-59·2) ml/kg/min, respectively), but was significantly lower in group 2 (27·7 (13·9-44·3) ml/kg/min) and group 3 (26·8 (22·6-36·7) ml/kg/min). The difference in maximal oxygen uptake between groups 1 and 2 was 10·8 ml/kg/min (95% confidence interval 3·6 to 23·4 ml/kg/min) and between groups 1 and 3, 11·7 ml/kg/min (4·9 to 22·5 ml/kg/min). These differences were not explained by differences in metabolic control or the degree of autonomic neuropathy.Thus the insulin dependent diabetics with only slightly increased urinary albumin excretion had an appreciably impaired aerobic work capacity which could not be explained by autonomic neuropathy or the duration of diabetes. Whether the reduced capacity is due to widespread microangiopathy or another pathological process affecting the myocardium remains to be established.     

Response of Asthmatics to Isoprenaline and Salbutamol Aerosols Administered by Intermittent Positive-pressure Ventilation

The bronchodilator and cardiac effects produced by aerosols of 0·5% isoprenaline and of 0·25, 0·5, and 1% salbutamol administered in 40% oxygen by intermittent positive-pressure ventilation were compared in 24 asthmatic patients. Isoprenaline and salbutamol in concentrations of 0·5% were equipotent in peak bronchodilator effect; salbutamol was superior in total bronchodilator effect and duration of average effect, but the peak bronchodilator effect occurred earlier after isoprenaline. Significantly greater tachycardia was produced by 0·5% isoprenaline than by the same concentration of salbutamol. The 0·25, 0·5, and 1% concentrations of salbutamol had about the same peak bronchodilator effect, but there was a stepwise increase in total effect and duration of average effect in relation to the concentration used. A similar stepwise increase in heart rate was also noted, but with all concentrations this was significantly less than with 0·5% isoprenaline. It was concluded that a 0·5% solution of salbutamol, which provided maximal bronchodilatation without important tachycardia, was therapeutically superior to the other three treatments.     

Interactions between glycosaminoglycans and proteins, with particular reference to a new technique for isolating serum glycoproteins

1. Chondroitin sulphate–serum protein complexes (A, B and C), successively precipitated by adding chondroitin sulphate to serum at three arbitrary descending pH values (5·2, 4·3 and 3·1), were dissociated at pH 6·7 and chromatographed on DEAE-Sephadex, when the liberated serum proteins were simultaneously freed of chondroitin sulphate and separated into five fractions. Evidence that serum proteins were precipitated as a result of electrostatic interactions with dissociated carboxylate groups on the glycosaminoglycan is presented. 2. Serum proteins (fraction G), unable to form complexes with chondroitin sulphate, contained 4·4% of sialic acid and accounted for 4 and 26% of the total protein and protein-bound sialic acid in serum respectively. This fraction interacted electrostatically with chondroitin sulphate only when rendered more basic by removal of sialic acid residues with neuraminidase. The heat stability, solubility properties and high carbohydrate content of fraction G classified it as a seromucoid fraction. 3. Fraction G contained several glycoprotein and hexuronic acid-containing fractions, including a hitherto undetected brown-pigmented high-molecular-weight serum component, which migrated in starch gel between the origin and Sα₂-globulin and contained 3·1 and 4·1% of sialic acid and hexuronic acid respectively. 4. Glycosaminoglycan–protein interactions are discussed in relation to protein fractionation. By prior removal of less acidic proteins by these interactions, a new technique is available for isolating serum seromucoids in higher yields and under milder conditions than existing methods.     

Rat-kidney lysosomes: isolation and properties

1. The activities of lysosomal enzymes in the cortexes and medullas and the principal subcellular fractions of rat kidney were measured. 2. A method is described for the isolation of rat-kidney lysosomes and a detailed analysis of the enzymic composition of the lysosomes is reported. Enzyme analysis of the other principal subcellular fractions is included for comparison. 3. Studies of the distribution of α-glucosidase showed that the lysosomal fraction contained only 10% of the total enzyme activity. The microsomal fraction contained most of the particulate α-glucosidase. Lysozyme was concentrated mainly in the lysosomal fraction with only small amounts present in the microsomal fraction. Lysosomal α-glucosidase had optimum pH5 whereas the microsomal form had optimum pH6. Both lysosomal and microsomal lysozyme had optimum pH6·2. 4. The stability of lysosomal suspensions was studied. Incubation at 37° and pH7 resulted in first an increased availability of enzymes without parallel release of enzyme. This was followed by a second stage during which the availability of enzymes was closely related to the release of enzymes. These changes were closely paralleled by changes in light-scattering properties of lysosomes. 5. The latent nature of the α-glucosidase and lysozyme of intact kidney lysosomes was demonstrated by their graded and parallel release with other typical lysosomal enzymes. 6. Isolated lysosomes were unstable at pH values lower than 5, most stable at pH6–7 and less stable at pH 8–9. Lysosomes were not disrupted when the osmolarity of the suspending medium was decreased from 0·6m to 0·25m. 7. The discussion compares the properties and composition of kidney lysosomes, liver lysosomes and the granules of macrophages. 8. The possible origin of the lysozyme in kidney lysosomes by reabsorption of the lysozyme in blood is discussed.     

Mechanisms of lipid peroxide formation in animal tissues

1. Homogenates of rat liver, spleen, heart and kidney form lipid peroxides when incubated in vitro and actively catalyse peroxide formation in emulsions of linoleic acid or linolenic acid. 2. In liver, catalytic activity is distributed throughout the nuclear, mitochondrial and microsomal fractions and is present in the 100000g supernatant. Activity is weak in the nuclear fraction. 3. Dilute (0·5%, w/v) homogenates catalyse peroxidation over the range pH5·0–8·0 but concentrated (5%, w/v) homogenates inhibit peroxidation and destroy peroxide if the solution is more alkaline than pH7·0. 4. Ascorbic acid increases the rate of peroxidation of unsaturated fatty acids catalysed by whole homogenates of liver, heart, kidney and spleen at pH6·0 but not at pH7·4. 5. Catalysis of peroxidation of unsaturated fatty acids by the mitochondrial and microsomal fractions of liver is inhibited by ascorbic acid at pH7·4 but the activity of the supernatant fraction is enhanced. 6. Inorganic iron or ferritin are active catalysts in the presence of ascorbic acid. 7. Lipid peroxide formation in linoleic acid or linolenic acid emulsions catalysed by tissue homogenates is partially inhibited by EDTA but stimulated by o-phenanthroline. 8. Cysteine or glutathione (1mm) inhibits peroxide formation catalysed by whole homogenates, mitochondria or haemoprotein. Inhibition increases with increase of pH.     

Stimulation of adenine phosphoribosyltransferase by adenosine triphosphate and other nucleoside triphosphates

1. Adenine phosphoribosyltransferase was protected from inactivation on heating at 55° by the presence of 5-phosphoribosyl pyrophosphate. ATP, adenine, AMP or GMP had no protective effect on the activity of this enzyme. The presence of either 5-phosphoribosyl pyrophosphate or ATP did not protect adenine phosphoribosyltransferase against the loss of ATP stimulation obtained by heating at 55°. 2. At pH5·3 and 6·0 adenine phosphoribosyltransferase was stimulated by a narrow range of ATP concentration (15–25μm). At pH6·5 and 7·0 maximum stimulation was obtained with 25–30μm-ATP, and at pH7·4, 8·2 and 8·85 maximum stimulation was obtained over a wide range of ATP concentrations (60–200μm). With extracts that had been heated for 30min. at 55° no stimulation was observed at either pH5·3 or 7·4 with ATP concentrations up to 100μm. 3. Short periods of heating at 55° (1, 2 or 5min.) increased the stimulation of adenine phosphoribosyltransferase obtained with various concentrations of ATP. 4. The addition of CTP, GTP, deoxy-GTP, deoxy-TTP or XTP to assay mixtures resulted in weak stimulation of adenine-phosphoribosyltransferase activity. 5. It is suggested that there are at least three different forms of adenine phosphoribosyltransferase, each with a different affinity for ATP.     

The isolation of neurophysin-I and -II from bovine pituitary neurosecretory granules separated on a large scale from other subcellular organelles. Demonstration of slow equilibration of neurosecretory granules during centrifugation in a sucrose density gradient

1. An improved procedure for the isolation of neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. 2. Of the total oxytocic and pressor activities present in the original tissue 80% was sedimentable. 3. The granules were separated from mitochondria by prolonged centrifugation in a sucrose density gradient. During a sedimentation period of 5hr. the granules moved progressively into denser regions of the gradient and the mitochondria remained at the top. 4. The biological activities of the granules were measured: the oxytocic activity was 11·56±1·63 and the pressor activity was 15·60±3·91 units/mg. of protein. 5. A protein was isolated from a lysate of granules prepared from 40 pituitary glands. Amino acid analysis showed that it consisted of a mixture of neurophysin-I and neurophysin-II in equal proportions. It accounted for 60% of the soluble granule protein and for 50% of the total granule protein. 6. The neurophysins present in the granules are associated with 19·1 units of oxytocic and 21·1 units of pressor activity/mg. of protein. 7. Starch-gel electrophoresis revealed the presence of both neurophysins in extracts of 15 pituitary glands studied individually. 8. We conclude that the polypeptide hormones, oxytocin and [8-arginine]-vasopressin, are normally closely associated with the two neurophysins within neurosecretory granules of the pituitary gland.     

Asparaginase and asparagine transaminase in soybean leaves and root nodules

Asparaginase activity (≤1 μmol/mg protein · hr) was detected in extracts of soybean (Glycine max [L.] Merr.) leaf blades, but, even after efforts to optimize extraction and assay of the enzyme, specific activity was not sufficient to metabolize the estimated amount of asparagine translocated to leaves. Asparagine transaminase activity with glyoxylate or pyruvate was at least 52 and 62 nmol/mg protein · hr, respectively. This estimate of transaminase activity is based on the analysis of the reaction product α-ketosuccinamate. Formation of glycine and alanine was confirmed by amino acid analysis. α-Ketosuccinamate deamidase had a specific activity of 85 nmol/mg protein · hr in leaf blade extracts.     

The purification and some properties of the polyphenol oxidase from tea (Camellia sinensis L.)

1. Polyphenol oxidase (EC 1. 10. 3.–) from the shoots of the tea plant was purified about 5000-fold on a dry-weight basis. 2. At an intermediate stage of purification four soluble yellow fractions were obtained. They are believed to represent complexes of a basic enzyme protein with acidic phenolic oxidation products and nucleic acids. After removal of the complex-forming materials the fractions were blue and similar to each other. About 40% of the activity could not be extracted from the acetone-dried powder. 3. Each of the four blue fractions was resolved further into two species, A and B. The following results refer to species A. 4. The enzyme showed absorption maxima at 279mμ (E^1%_1cm., 13·5) and 611mμ (E^1%_1cm., 0·84) with a shoulder at 330mμ. The enzyme was bleached by substrate under anaerobic conditions and the colour was restored by oxygen. 5. The molecular weight measured by sedimentation and diffusion was 144000±16000. The copper content was 0·32% (w/w). 6. Kinetic constants are given for a number of substrates and inhibitors, including the natural substrates of the tea leaf. The specific activity towards pyrogallol was 373 units/mg. at 30°. 7. The best substrates were o-dihydric phenols. Quinol and p-phenylenediamine were slowly oxidized. Monohydric phenols and ascorbic acid were not oxidized. 8. The kinetics of oxidation of most substrates are consistent with a mechanism in which oxidized and reduced forms of the enzyme form binary complexes with phenol and oxygen respectively. A modified mechanism is postulated for the oxidation of chlorogenic acid. 9. The relation of the results to the mechanism of tea fermentation is discussed.     

The isolation and properties of epithelial-cell `ghosts' from rat small intestine

1. The preparation of gram quantities of isolated epithelial-cell `ghosts'' from mucosal scrapings of rat small intestine is described. The method involves dispersing the tissue by gentle homogenization in 6% dextran in Krebs–Ringer phosphate, pH7·4, followed by filtration through nylon cloth and sedimentation by low-speed centrifuging. 2. The isolated epithelial-cell `ghosts'' contained all of the DNA, but only 52% of the protein and 53–57% of the RNA of the original homogenate. They contained most of the activity of the following enzymes found in the homogenate: aminopeptidase (71%); alkaline β-glycerophosphatase (82%); invertase (92%); adenosine triphosphatase (93–116%); acid β-glycerophosphatase (83%); nonspecific esterase (76%); succinate dehydrogenase (96%). Only small proportions of the total lactate-dehydrogenase (10%) and phosphoglucose-isomerase (2%) activities found in the homogenate were recovered in the isolated cell `ghosts''. 3. The epithelial-cell `ghost'' preparation did not respire unless cofactors and substrates were added, and did not consume glucose or produce lactic acid from glucose. 4. The effect of varying the composition of the homogenization medium was studied. Concentrations of dextran (mol.wt. 15×10⁴) from 1 to 12%, solutions of dextrans (all at 6%) with mol.wt. varying between 3·6×10⁴ and 2×10⁶, and a solution of 8% polyethylene glycol (mol.wt. 4000) served equally well for the production of epithelial-cell `ghosts''. Two of these solutions, however, 12% dextran (mol.wt.15×10<sup>4</sup>) and 6% dextran (mol.wt. 2×10<sup>6</sup>), were too viscous to allow the complete sedimentation of the cell `ghosts'' at low relative centrifugal forces. Omission of either Krebs–Ringer phosphate or dextran from the medium resulted in almost complete cell breakage during the homogenization. 5. The isolated cell `ghosts'' were used as a starting material for subcellular fractionation of rat intestinal mucosa by differential centrifugation. The distributions of protein and succinate-dehydrogenase activity among the fractions were compared with corresponding values in fractions isolated by differential centrifugation of mucosa homogenized in 0·3m-sucrose–5mm-EDTA, pH7·4. The method in which cell `ghosts'' were used as starting material gave a better separation and cleaner fractions than the method in which untreated mucosal scrapings were used.     

PANCREATIC MICROSOMES : AN INTEGRATED MORPHOLOGICAL AND BIOCHEMICAL STUDY

The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, (~15 mµ), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mµ), limited by a thin membrane (7 mµ) which bears small (~15 mµ) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules (~150 mµ) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small (~15 mµ), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.     

Ribonucleic acids from barley leaves

1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The T_m (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas T_m of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition, and the RNA from the supernatant fraction in accordance with its high T_m had a high GMP+CMP content.     

Factors protective against retinopathy in insulin-dependent diabetics free of retinopathy for 30 years

To investigate which factors might protect against the development of retinopathy 40 insulin-dependent diabetics who had remained free from retinopathy despite diabetes of long duration (mean±1 SD 30±10 years) were compared with 40 patients who had background and 47 who had proliferative retinopathy (mean durations of disease 16±5 and 19±5 years respectively). The three groups had had similar mean ages at onset of diabetes. The mean of all postprandial blood glucose measurements at hospital clinics from diagnosis of diabetes to detection of retinopathy, or to the most recent negative eye examination, was 9·9±2·1 mmol/l (178±38 mg/100 ml) in the group with no retinopathy, 11·8±2·1 mmol/l (213±38 mg/100 ml) in those with background retinopathy, and 12·4±2·1 mmol/l (223±38 mg/100 ml) in those with proliferative retinopathy (p <0·0001). This difference was not reflected in present concentrations of haemoglobin A_1C, probably because glycaemic control had been improved after the development of retinopathy. In the groups with background and proliferative retinopathy there were significant negative correlations between mean blood glucose concentrations and the number of years that had elapsed from diagnosis of diabetes to detection of retinopathy, suggesting that the development of both grades of retinopathy depends on the degree and duration of glycaemic exposure.The patients with no retinopathy had attended clinic more frequently (p <0·025), more of them had required emergency hospital treatment for hypoglycaemia (p <0·0025), and they tended to have had a lower prevalence of hyperglycaemic coma than the other groups. Although mean percentage ideal body weight and diastolic blood pressure were lower in the patients with no retinopathy at the time of study, mean body weight, blood pressure, and the prevalence of smoking in the years before the development of retinopathy had been similar in all groups, suggesting that these did not influence the development of retinopathy.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.     

The isolation of the native hormone-binding proteins from bovine pituitary posterior lobes. Crystallization of neurophysin-I and -II as complexes with [8-arginine]-vasopressin

1. The native hormone-binding proteins, neurophysin-I and -II, have been isolated from acetone-desiccated bovine pituitary posterior lobes. 2. Neurophysin-I and -II are present in approximately equal quantities in the tissue and are localized in the neurosecretory granules. 3. The apparent molecular weight, determined by equilibrium sedimentation of neurophysin-I, was 19000 and that of neurophysin-II was 21000; their sedimentation coefficients, S_20,w, were 1·66 and 2·02s respectively. 4. Neurophysin-I and -II are similar in amino acid composition. Neurophysin-II was distinguished from neurophysin-I by the absence of histidine. 5. The proteins form complexes with oxytocin as well as with vasopressin. Complexes of both proteins with [8-arginine]-vasopressin have been crystallized. 6. Bioassay of the pressor and oxytocic activities of the crystals shows that neurophysin-I binds three molecules of either vasopressin or oxytocin whereas neurophysin-II binds only two molecules of each hormone per molecule of protein. Complexes containing two molecules of oxytocin and one molecule of [8-arginine]-vasopressin per molecule of protein are formed by neurophysin-I and -II; both proteins appear to possess three polypeptide-binding sites/molecule.     

Response to Rimiterol and Salbutamol Aerosols Administered by Intermittent Positive-pressure Ventilation

The bronchodilator and cardiac effects produced by aerosols of 0·5% salbutamol and 0·5% and 1% rimiterol administered for three minutes in 40% oxygen by intermittent positive-pressure ventilation (I.P.P.V.) were compared in 15 asthmatic patients. Salbutamol and both the concentrations of rimiterol were equipotent in peak bronchodilator effect, but salbutamol had a significantly longer duration of bronchodilator action. There was significantly less increase in heart rate after rimiterol than after salbutamol. Aerosols of 0·5% rimiterol, 0·5% salbutamol, and saline were administered by I.P.P.V. to 10 normal volunteers. There was no difference between the mean heart rates after 0·5% rimiterol and saline but a highly significant increase in mean heart rate was observed after 0·5% salbutamol. It was concluded that 0·5% rimiterol was an effective short-acting bronchodilator drug with little or no cardiac beta₁-adrenergic activity when administered for three minutes by I.P.P.V. in 40% oxygen.     

ELECTRON MICROSCOPY OF LYSOSOME-RICH FRACTIONS FROM RAT THYMUS ISOLATED BY DENSITY-GRADIENT CENTRIFUGATION BEFORE AND AFTER WHOLE-BODY X-IRRADIATION

Fractions from rat thymuses were isolated by sucrose density-gradient centrifugation, before and after 1000 r whole-body x-irradiation, and examined by electron microscopy. Cytochrome oxidase and acid phosphatase activities of these fractions were tested as well. Electron-opaque bodies with diameters ranging from 0.10 to 0.35 µ, with a mean of 0.25 µ, were found in fractions having high acid phosphatase activity, while the fractions rich in cytochrome oxidase consisted mostly of mitochondria. After irradiation, there was an increased ratio of dense bodies to mitochondria. These particles are considered to be lysosomes similar to those identified in other rat tissues. Their relationship to the mitochondria is discussed.     

Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase

1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10·5) extraction of cytokinase from rabbit kidney lysosome–microsome fraction, followed by chromatography on DEAE-cellulose at pH7·6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. 3. Cytokinase is a protein with E^0·1%_1cm.=0·87 at 280mμ, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S_20,w 2·9–3·1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8·5. It is inactivated by heat at 78°. 4. Cytokinase and human urokinase have the same K_m value and are inhibited in a partially competitive manner by -aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.