THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

ON THE MECHANISM OF MILK SECRETION : THE INFLUENCE OF INSULIN AND PHLORIDZIN

The four types of experiments on milk secretion herein described really fall into one general class so far as the physiological effects produced are concerned. Starvation lowers the blood sugar and raises the osmotic pressure of the blood. The experiment using parathyroid hormone with or without starvation may have its effects interpreted as simply due to starvation since 1000 units of this hormone produced no visible effects on the blood calcium or milk constituents different from those of starvation. Since insulin produces a marked and rapid drop in blood sugar it too may be looked upon as a rapid starvation effect. It has some other important effects, however. Briggs et al. (21) have shown that potassium and phosphorus of the blood are decreased and Luck, Morrison, and Wilbur (22) indicate a reduction in the amino acids of the blood in insulin treatment. Phloridzin lowers the threshold for sugar retention with the consequence that in time it tends to lower the sugar of the blood to an even greater extent than that noted in starvation. It tends to depress the potassium, to increase the phosphorus content of the blood, and to cause the body to burn protein rather than carbohydrate, thus increasing nitrogen excretion. All of the experiments are characterized by a sharp reduction in the milk yield. Cary and Meigs (23) have studied like reductions in milk yield produced by varying the energy or protein of the diet. They conclude that such decrease in milk production may be interpreted as due to the direct effect of the starvation and the consequent reduction of the energy and protein available to milk secretion. The reduction in milk yield for the experiments herein described can undoubtedly be attributed to the same causes as those cited by Cary and Meigs. The experiment where Cow 47 was given a full ration and at the same time injected with large quantities of insulin is of particular interest in this connection. The ration was adequate and the cow ate well, yet her production declined to a fifth of her normal milk yield. Her chart shows that there was a slight reduction in her blood sugar when insulin was introduced into the blood stream. It seems furthermore likely that this sugar was not as available to milk secretion, since there appears to be more than a corresponding drop in the lactose content of the milk. The work of Luck et al. would seem to indicate that there should be a like drop in the amino acids of the blood. These two conditions would lead, according to the work of Cary and Meigs, to a reduction in the concentration of the nitrogen of the milk. Actually, in the experiment as it was performed, the nitrogen increased to a value about 40 per cent above normal. A somewhat similar conflict is noted in two of the other three insulin experiments where starvation accompanied insulin injection. To this extent it would seem that the factor deserving most emphasis in its immediate effect on milk yield is the energy available, and that the later and more secondary factor is the amino acid concentration of the blood. In the starvation experiments, the butter fat percentage of the milk rises rather uniformly with the duration of starvation. In the insulin experiments, however, the charts appear to show a marked reduction in this butter fat percentage immediately after the introduction of insulin. This is particularly noticed after the second and third injections. Since the dextrose of the blood tends to be reduced and made unavailable to the general physiological processes by the presence of the large excess of insulin, and since this reduction of the butter fat percentage is noted as an accompanying phenomenon, it would appear that the blood dextrose plays a part in the synthesis of milk fat as well as being the source of the milk lactose, possibly as a source of energy in converting body fat to butter fat. In this regard the results for the treatment of Cow 47 with phloridzin are of importance. As noted by others, the introduction of phloridzin causes a marked rise in the fat percentage of the milk. The lactose per cent is also higher than that noted in starvation. Since phloridzin, by lowering the threshold for the blood sugar, causes large quantities of it to be drained from the body through the urine, and therefore reduces the reserve supply, it follows that if the insulin hypotheses are correct we should expect an eventual lowering of the lactose and of the fat below the starvation level. During the last of the experiment this is what was actually observed. The effects of starvation and of insulin furnish concordant proof for the theory that the lactose of milk is derived from the sugar of the blood. The fact that the different constituents of the milk, the fat, the lactose, the nitrogen, and the ash, do not exactly parallel each other in their behavior throughout these experiments indicates that they have in all probability separate origin. This is particularly true of the butter fat percentage, which appears to have a rate of secretion which is more or less independent of the other constituents, and higher in amount. This result would fall in line with the conclusion of the writers in a previous paper in which it was indicated that the fat of the blood was very likely deposited in the udder as fat corresponding to body fat from which source it was metabolized into the fat of milk shortly before it was needed for milk secretion. The wide variation brought about in the constituents of the milk by the treatment all point to the conclusion that in milk secretion a balance is maintained between the osmotic pressure of the milk and of the blood. Thus when the sugar of the milk is reduced either through starvation or by insulin the ash constituents rise to compensate for this reduction and make the osmotic pressure of the milk similar to that of the blood. These results further appear to indicate that the salts and the sugars are more or less independent in their passage and metabolism into milk from the other constituents. These observations are therefore in line with those obtained by Jackson and Rothera (14) and by Davidson (15) in their brilliant experiments where they modified milk secretion by returning milk or milk sugars and salts to the udder. These experiments give direct proof for the conclusion that modifications of the blood of dairy cattle produce direct and predictable modification of the milk secreted.     

AN ATTEMPT AT PEPTIC SYNTHESIS OF INSULIN

1. Synthesis of plastein from the products of peptic hydrolysis of small quantities of egg albumin can be demonstrated with amorphous or crystalline pepsin. 2. Synthesis of plastein from the products of peptic hydrolysis of amorphous or crystalline insulin can be demonstrated with amorphous or crystalline pepsin. 3. The plastein synthesised by pepsin from the products of peptic hydrolysis of insulin is physiologically inactive. 4. The plastein formed in the insulin experiments could not be crystallised by the methods used for the crystallisation of insulin. 5. The physiological activity of insulin is not destroyed by repeated freezing (at about –50°C.) and melting of an aqueous or an alcoholic solution of this hormone. 6. No marked decrease in the physiological activity of insulin after incubation at 37°C. with pepsin at pH 4.0, in dilute or concentrated solutions, was detected.     

ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-¹⁴C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-¹⁴C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.     

FRACTIONATION OF OLFACTORY TISSUE HOMOGENATES. ISOLATION OF A CONCENTRATED PLASMA MEMBRANE FRACTION

Abstract— Differential and sucrose-density-gradient centrifugation techniques were used for studies on the separation of subcellular particles from rabbit brain and olfactory tissue. Comparisons were made among various fractions from the two types of tissue. These comparisons included protein concentration and enzyme activities of the individual fractions as well as their distribution in subfractions from density gradient separations. In tissue whole homogenates, the percentage of total ATPase activity as ouabain sensitive Na⁺-K⁺ ATPase activity was about 4 times greater in brain cortex (63 per cent) than in olfactory tissue (17 per cent). Cytochrome oxidase and Na⁺-K⁺ ATPase activities were used to indicate the presence and the concentration of mitochondria and of the plasma membranes. A fraction with properties similar to the mitochondria plus nerve ending fraction from brain homogenates (fraction B) was obtained from olfactory tissue. Nerve ending concentration subfractions (B₂) were prepared from the B primary fractions. Plasma membrane subfractions were obtained by osmotic shock treatment of B₂, In the fraction of plasma membrane from olfactory tissue (E₂), 56 per cent of the total ATPase activity was Na⁺-K⁺ ATPase activity. In E₂ from brain 71 per cent was Na⁺-K⁺ ATPase activity. Deoxycholate (DOC)-treated fractions containing nerve endings from brain preparations showed much greater increase in cytochrome oxidase activity than did similar fractions from olfactory tissue. DOC treatment increased the NADH cytochrome c reductase activity of all fractions and subfractions from brain, while it decreased activity in all but one fraction from olfactory tissue. DOC treatment decreased both the Mg²⁺ and Na⁺-K⁺ ATPase activities in both types of tissue. Electron photomicrographs of olfactory B₂, B₃, E₂ and E₃ show clear morphological differences among these subfractions. The presence of possible cilia and basal bodies on vesicles in B₂ gives morphological evidence for the presence of terminal swellings in this subtraction in agreement with enzyme marker activity results.     

THE INFLUENCE OF COUMESTROL,ZEARALENONE, AND GENISTEIN ADMINISTRATION ON INSULIN RECEPTORS AND INSULIN SECRETION IN OVARIECTOMIZED RATS

ABSTRACT

The influence of phytoestrogens (genistein and coumestrol) and mycoestrogen (zearalenone) on insulin secretion, liver insulin receptors and some aspects of lipid and carbohydrate metabolism were investigated in this study. Ovariectomized rats were injected s.c. with the above mentioned compounds in the amount of 1?mg for three days. Coumestrol and zearalenone caused a significant increase in uterus weight, similar to the effects observed after estrone action, while this effect was not observed after the genistein injection. Blood insulin level was not changed after phyto- or mycoestrogen treatment. However, coumestrol and genistein significantly decreased the binding capacity of liver insulin receptors. These changes corresponded with alterations in glucose and free fatty acids profiles in blood, as well as with glycogen content in liver. The effects observed after genistein and coumestrol injections differed from those noticed in rats treated with zearalenone or estrone. On the basis of these results we conclude that metabolic effects of high doses of coumestrol and genistein in ovariectomized rats are partly mediated by changes in insulin sensitivity of the liver and that the action of plant estrogens on metabolism is, at least to the some degree, independent of their estrogen activity.     

AN ECOPHYSIOLOGICAL STUDY OF THE SALT SECRETION OF FOUR HALOPHYTES

Plants of Spartina anglica, Limonium vulgare, Armeria maritima and Glaux maritima were collected in the field and grown on different concentrations of NaCl, KCl and CaCl₂. Salt secretion, ion content, water content and transpiration rates were determined. The highest sodium secretion was found in Spartina anglica , a species from the most saline habitat; and a somewhat lower secretion rate in Limonium vulgare. The lowest rates were found in Glaux maritima and Armeria maritima. The sodium secretion efficiency, i.e. the ability to maintain an unchanged internal sodium content, was highest in Spartina anglica. Spartina anglica is the most successful in the removal of excessively absorbed salt, since it secretes 60% of the absorbed sodium. The values for Limonium vulgare, Glaux maritima and Armeria maritima were 33, 20 and 4% respectively. The species studied differ in the preferential sequence of ion secretion as well as in secretion rate and efficiency. This preferential sequence of ion secretion seems to be similar in members of the same taxonomic group (Plumbaginaceae). The comparability of the secretion parameters is discussed with regard to morphological differences between the species.     

THE CEREBELLAR GLOMERULUS: ISOLATION AND METABOLIC PROPERTIES OF A PURIFIED FRACTION

Abstract— A method is described for large scale isolation of glomerular complexes from rabbit cerebellum. The purity of the fraction is 90–95%, measured by quantitative electron microscopy. In addition biochemical markers indicate a high degree of particle integrity. The glomeruli occur as mechanically separable units at 20–40 days of animal age and the amount of protein per particle is 15–20 × 10⁻¹¹ g. The glomeruli accumulate [³H]GABA, and exhibit both high ( K _m 15μM) and low ( K _m 0.5 mM) affinity uptake properties. Glomeruli oxidize α-glycerophosphate and succinate particularly well, while glutamate, pyruvate and α-ketoglutarate are less effective as respiratory substrates.     

EFFECT OF AN OXYGEN-REDUCING MEMBRANE FRACTION ON HELICOBACTER PYLORI

The emerging gastric pathogen Helicobacter pylori is an oxygen-sensitive fastidious microaerophile. Culturability of this organism is rapidly lost in oxygen levels present in the atmosphere due to its morphological transformation into a viable but not culturable state. The effect of the Oxyrase^TM system of oxygen-reducing membrane fragments on H. pylori was evaluated at levels ranging from 0.1 to 0.6 Units/mL in Brucella broth supplemented with 5% horse serum. Duplicate sets of Oxyrase^TM dilutions inoculated with H. pylori were incubated at 35C aerobically and microaerobically. At these Oxyrase^TM levels, a logarithmic loss of H. pylori viability was evident in the aerobic cultures. The inoculum remained recoverable for 24 h in the presence of Oxyrase^TM, whereas recovery of inoculum in untreated broth was greatly reduced after 8 h of aerobic incubation, and the organism was unrecoverable after 24 h. Oxyrase^TM-containing broth cultures of H. pylori incubated microaerobically showed a similar drop in viable counts for the first 48 h of incubation; however, at the lower levels of Oxyrase^TM, some cells survived, and resumed logarithmic growth at 96 h. To explore the effects of short term aerobic incubation in the presence of 0, 0.005, 0.05, and 0.5 Units Oxyrase^TM, cultures were examined microscopically after 4, 8, and 24 h. In the Oxyrase^TM-containing broths, <90% of the cells exhibited rod shape morphology after 8 h, whereas in the untreated broth, most cells appeared coccoid. After 24 h, all cells exhibited coccoid morphology.     

AN ANALYSIS OF COLLAGEN SECRETION BY ESTABLISHED MOUSE FIBROBLAST LINES

In vitro synthesis of collagen by established mouse fibroblast lines has been examined by electron microscopy. During rapid growth (log phase), when collagen could not be detected in the cultures, the cells lacked a well developed granular ergastoplasm and Golgi system. Upon cessation of growth (stationary phase), collagen accumulated in the cultures and the cells demonstrated highly developed granular and smooth ergastoplasm. Collagen appeared to be synthesized in the rough-surfaced endoplasmic reticulum and to be transported as a soluble protein to the cell surface by vesicular elements of the agranular ergastoplasm. Fusion of the limiting membranes of these vesicles with the cell membrane permitted the discharge of the soluble collagen into the extracellular space, where fibrils of two diameter distributions formed. The secretion of collagen is concluded to be of the merocrine type. Alternative theories of collagen secretion are discussed and the data for established lines compared with the results of other in vitro and in vivo studies of collagen fibrillogenesis.     

ISOLATION AND PROPERTIES OF AN EXOCELLULAR NUCLEASE OF SERRATIA MARCESCENS

Eaves, George N. (Wayne State University College of Medicine, Detroit, Mich.) and Charles D. Jeffries. Isolation and properties of an exocellular nuclease of Serratia marcescens. J. Bacteriol. 85:273-278. 1963.-The exocellular nuclease of Serratia marcescens, isolated by anion-exchange chromatography on diethylaminoethyl-Sephadex, depolymerized deoxyribonucleic acid, ribonucleic acid, and the polynucleotide which is refractory to pancreatic ribonuclease activity. The enzyme was tentatively classified as a nonspecific phosphodiesterase. Magnesium was essential for activity, which was optimal at pH 8.8. The purified enzyme was completely inactivated by heating at 50 C for 15 min.     

ANALYSIS OF RNA SYNTHESIZED BY AN ISOLATED RAT BRAIN SYNAPTOSOMAL FRACTION

Abstract— A synaptosomal fraction derived from rat brain, when incubated in an appropriate medium. incorporates [5-³H]uridine into RNA. On the basis of sedimentation analysis, RNA-DNA hybridization and metabolic inhibitor studies, mitochondrial RNA species appear to be the major, if not sole, RNA products synthesized by the isolated synaptosomal fraction. Electronmicroscope autoradiographic analysis showed that about 50% of the incorporation of [5-³H]uridine into RNA by this fraction occurs in the presynaptic endings. The capacity of mitochondrial RNA synthesis in isolated nerve endings declines as the age of the animal increases from 10 to 30 days, and by 60 days of age discrete mitochondrial RNA species are barely detectable.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

CHARACTERIZATION OF THE FRACTION OBTAINED FROM THE CNS OF JIMPY MICE BY A PROCEDURE FOR MYELIN ISOLATION

Abstract— A subcellular fraction (called the 0·85-fraction) was isolated from the brains of Jimpy mice by a procedure for obtaining myelin of high purity from immature normal brains. The yield of this fraction obtained from 17-day-old Jimpy mice was only 5 per cent of that from age matched controls. In the electron microscope, the O·85-fractions obtained from 9- and 17-day-old control mice showed many multilayered whorls of myelin, whereas the corresponding fraction from the Jimpy mice was free of multilayered structures which could be recognized as myelin. Basic proteins, proteolipid protein and galactocerebrosides could not be detected in the 0·85-fraction from Jimpy mice although they were major components of the 0·85-fractions from both 9- and 17-day-old control mice. The specific activity of 2',3'-cyclic nucleotide 3'- phosphohydrolase in the Jimpy 0·85-fraction was only 15 per cent of the value for controls. These results can be explained either by the 0·85-fraction from Jimpy brain being a very abnormal 'myelin' or by its being primarily non-myelin contaminants. Little or none of the major glycoprotein found in normal myelin fractions was found in the 0·85-fraction from Jimpy brains. This finding is strong evidence indicating that the glycoprotein is closely associated with normal myelin in situ.     

DEVELOPMENT OF AN IMPROVED MEDIUM FOR THE ISOLATION OF LIVER MITOCHONDRIA

In view of the unsatisfactory appearance, under the electron microscope, of liver mitochondria isolated in isotonic sucrose medium, alternative media have been examined. It was found to be advantageous to replace sucrose by raffinose, and to add levan or, preferably, dextran, together with heparin in suitable concentration. With the optimal medium, the constituents of which are raffinose, versene (optional), dextran of high molecular weight, heparin, and AMP (optional), most of the mitochondria in the osmium-fixed pellet are apparently intact, and show the membranes characteristic of mitochondria as seen in cell sections. The optimal medium has no adverse effect on the activity of the several tissue enzymes which have been studied, except that Mg⁺⁺-activated ATPase is partially inhibited if the medium is present in high concentration in the assay system. Mitochondrial fractions isolated in the new medium have, in common with sucrose fractions, appreciable "free" ATPase activity, this activity being evidently a poor criterion of mitochondrial integrity. Use of the new medium does not decrease the proportion of cytoplasmic ATPase which fails to sediment with the mitochondria, but does give a mitochondrial fraction low in RNA and in acid phosphatase activity and little contaminated with microsomal material. Particles tentatively identified as "lysosomes" have been seen in certain sections.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : I. Method and Morphology

Golgi apparatus were released without fixatives from rat hepatocytes by gentle homogenization, concentrated by differential centrifugation, and purified by sucrose gradient centrifugation. Examination of sections of purified fractions by electron microscopy showed fields of morphologically intact units of Golgi apparatus consisting of stacks of parallel flattened cisternae, secretory vesicles, and small vesicular profiles. Negative staining of unfixed pellets revealed a complex network of anastomotic tubules continuous with platelike structures and secretory vesicles. These structures corresponded, respectively, to the small vesicular profiles and parallel flattened cisternae with attached secretory vesicles of sectioned material. Small fragments of granular endoplasmic reticulum were often closely associated with the peripheral tubules, suggesting sites of continuity in intact hepatocytes.     

AN ISOLATION UNIT FOR SUPPLYING RADIOISOTOPES TO SPECIFIC SEGMENTS OF AN INTACT ROOT

A device useful for supplying radioactive ions to specific segments of intact roots is described. A small volume of aerated, radioactive culture solution may be circulated around an isolated segment of a root, while the remainder of that root and the other roots of the plant are exposed to non-radioactive culture solution. Any root which does not have lateral roots extending beyond the epidermis can be inserted into the device without handling the root itself. No sealants are required to achieve a leak-proof seal above and below the root segment. The use of flexible rubber membranes to isolate root segments causes no detectable damage to root cells where the membranes contact the root epidermis.     

蜚蠊灭菌肽的诱导及初步分离分析

昆虫经诱导盾产生灭菌肽的研究近年来已有很大进展,有关这方面的研究工作绝大多数都是以有翅亚纲内生翅类鳞翅目(主要是蚕类)昆虫和少数双翅目昆虫为材料.本文首次以有翅亚纲外生翅类蜚蠊目的美洲蜚蠊(Ptriplaneta americana L.)为实验昆虫,用Escherichia coil K₁₂ strain D₃₁作诱导源,对不同发育期、不同性别、不同成虫期的蜚蠊进行诱导后,采用含菌培养基平板测活方法,就存在个体数及能产生抗菌物质的个体数进行了初步的研究.发现成虫日龄在10天之内的雄性蜚蠊能够产生抗菌物质的个体百分比最高.抗菌物质出现的高峰期是在诱导后第三、四天.用滴滴涕和溴氰菊酯作诱导源对雄性蜚蠊的诱导实验表明,杀虫剂也能诱导蜚蠊产生抗菌物质,而且所诱导产生抗菌物质的活性强度(用抑菌圈直径表示)高于大肠杆菌所诱导的.滴滴涕和溴氰菊酯的重复诱导可提高蜚蠊产生抗菌物质个体百分比.蜚蠊经诱导后产生的抗菌物质具有广谱性,对苏云金杆菌、金黄色葡萄球菌、枯草杆菌及绿脓杆菌等有较强的抗菌活性,而对大肠杆菌D₃₁、大肠杆菌、粘质沙雷氏杆菌和溶壁微球菌等有较弱的抗菌活性.用肽类物质的指纹图谱法分离蜚蠊血淋巴抗菌物质,发现经诱导后血淋巴中确有新的肽类物质产生,该物质具抗菌活性,用DABITC法分析,其N-末端氨基酸为赖氨酸.