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, ( approximately 15 mmicro), 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 mmicro), limited by a thin membrane (7 mmicro) which bears small ( approximately 15 mmicro) 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 ( approximately 150 mmicro) 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 ( approximately 15 mmicro), 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.     

LIVER MICROSOMES : AN INTEGRATED MORPHOLOGICAL AND BIOCHEMICAL STUDY

Rat liver, liver homogenates, and microsome fractions separated therefrom were examined systematically in the electron microscope in sections of OsO₄-fixed, methacrylate-embedded tissue and pellets. It was found that most microsomes are morphologically identical with the rough surfaced elements of the endoplasmic reticula of hepatic cells. They appear as isolated, membrane-bound vesicles, tubules, and cisternae which contain an apparently homogeneous material of noticeable density, and bear small, dense particles (100 to 150 A) attached to their outer aspect. In solutions of various osmolar concentrations they behave like osmometers. The findings suggest that they derive from the endoplasmic reticulum by a generalized pinching-off process rather than by mechanical fragmentation. The microsome fractions contain in addition relatively few vesicles free of attached particles, probably derived from the smooth surfaced parts of the endoplasmic reticula. Dense, peribiliary bodies represent a minor component of the same fractions. The microsomes derived from 1 gm. wet weight liver pulp contained (averages of 10 experiments) 3.09 mg. protein N, 3.46 mg. RNA (RNA/protein N = 1.12), and 487 µg. phospholipide P. They displayed DPNH-cytochrome c reductase activity and contained an alcohol-soluble hemochromogen. The microsome preparations proved resistant to washing and "aging." Treatment with versene and incubation with ribonuclease (30 minutes at 37°C.) resulted in appreciable losses of RNA and in partial or total disappearance of attached particles. Treatment with deoxycholate (0.3 to 0.5 per cent, pH = 7.5) induced a partial clarification of the microsome suspensions which, upon centrifugation, yielded a small pellet of conglomerated small, dense particles (100 to 150 A) with only occasionally interspersed vesicles. The pellet contained ~80 to 90 per cent of the RNA and ~20 per cent of the protein N of the original microsomes. The supernatant accounted satisfactorily for the materials lost during deoxycholate treatment. The findings suggest that the microsomal RNA is associated with the small particles whereas most of the protein and nearly all of the phospholipide, hemochromogen, and DPNH-cytochrome c reductase activity are associated with the membrane or content of the microsomes.     

Liver microsomes; an integrated morphological and biochemical study

Rat liver, liver homogenates, and microsome fractions separated therefrom were examined systematically in the electron microscope in sections of OsO(4)-fixed, methacrylate-embedded tissue and pellets. It was found that most microsomes are morphologically identical with the rough surfaced elements of the endoplasmic reticula of hepatic cells. They appear as isolated, membrane-bound vesicles, tubules, and cisternae which contain an apparently homogeneous material of noticeable density, and bear small, dense particles (100 to 150 A) attached to their outer aspect. In solutions of various osmolar concentrations they behave like osmometers. The findings suggest that they derive from the endoplasmic reticulum by a generalized pinching-off process rather than by mechanical fragmentation. The microsome fractions contain in addition relatively few vesicles free of attached particles, probably derived from the smooth surfaced parts of the endoplasmic reticula. Dense, peribiliary bodies represent a minor component of the same fractions. The microsomes derived from 1 gm. wet weight liver pulp contained (averages of 10 experiments) 3.09 mg. protein N, 3.46 mg. RNA (RNA/protein N = 1.12), and 487 microg. phospholipide P. They displayed DPNH-cytochrome c reductase activity and contained an alcohol-soluble hemochromogen. The microsome preparations proved resistant to washing and "aging." Treatment with versene and incubation with ribonuclease (30 minutes at 37 degrees C.) resulted in appreciable losses of RNA and in partial or total disappearance of attached particles. Treatment with deoxycholate (0.3 to 0.5 per cent, pH = 7.5) induced a partial clarification of the microsome suspensions which, upon centrifugation, yielded a small pellet of conglomerated small, dense particles (100 to 150 A) with only occasionally interspersed vesicles. The pellet contained approximately 80 to 90 per cent of the RNA and approximately 20 per cent of the protein N of the original microsomes. The supernatant accounted satisfactorily for the materials lost during deoxycholate treatment. The findings suggest that the microsomal RNA is associated with the small particles whereas most of the protein and nearly all of the phospholipide, hemochromogen, and DPNH-cytochrome c reductase activity are associated with the membrane or content of the microsomes.     

CYTOCHEMICAL STUDY ON THE PANCREAS OF THE GUINEA PIG : VII. Effects of Spermine on Ribosomes

Pancreatic ribosomes (guinea pig) aggregate and lose upon treatment with polyamines, particularly spermine, their bound secretory enzymes. Spermine, at 0.5 mM, for example, causes the release of about 85 per cent of the chymotrypsinogen and RNase, and from 85 to 100 per cent of the ribosomal amylase. At the same time, the particles lose about 10 per cent of their RNA, 7 to 24 per cent of their total protein, and from 75 to 100 per cent of their Mg⁺⁺. Observations with the electron microscope confirm the heavy agglutinating of the ribosomes but otherwise show little change in the structure of the particles. Using radioactive spermine it was found that, concomitant with the loss of bound enzymes and Mg⁺⁺ from the ribosomes, spermine became bound to the particle. The extent of binding ranged from 0.29 to 1.49 µmoles per 10µmoles RNA-P. The bound radioactive spermine can be removed by subsequent treatment of the ribosomes with GTP, ATP, or P-P, which treatment also removes most of the RNA of the particles, leaving behind ribosomes with a much lower RNA/protein ratio. From this evidence it was inferred that spermine, in releasing the Mg⁺⁺ of the particle, becomes salt-linked to the free phosphate hydroxyl groups of the RNA. Freshly isolated pancreatic and hepatic ribosomes contain very little spermine, about 0.1 to 0.2 µmoles polyamine/10 µmoles RNA-P. The results are discussed in terms of the linkages between the structural protein, the bound secretory enzymes, and the RNA of the ribosomes.     

ELECTRON MICROSCOPE STUDIES OF RABIES VIRUS IN MOUSE BRAIN

The cells of brains of 2- and 3-day old mice infected with street rabies virus were examined in the electron microscope. It was observed that characteristic rod-like or elongated particles were found within a "matrix" in the cytoplasm of nerve cells and of astrocytes. These rod-like particles can be separated into two types, on the basis of slightly different morphological features. One particle is 110 to 120 mµ wide and has double-membraned coats; the other is 120 to 130 mµ wide and is covered by a single limiting membrane. The former is closely associated with the endoplasmic reticulum. The biological relationship between the two types is unknown, but both types of particles are considered to be street rabies viruses because of their structural features. It is believed that segmentation and branching of elongated particles may play a role in virus multiplication. Negri bodies appear as dense round bodies containing various coarse structures but no virus particles.     

ELECTRON MICROSCOPIC STUDY OF THE CYTOPATHOLOGY OF ECHO VIRUS INFECTION IN CULTIVATED CELLS

The cultivated monkey kidney cell is subject to changes when infected with ECHO viruses 6, 9, and 19. The electron microscope reveals three stages of infection: (a) initial stage. The nucleus appears granular with chromatin condensation on the nuclear envelope. The cytoplasm contains electron transparent vesicles and vacuoles forming nests. (b) Intermediate stage. The nucleus seems to diminish, appearing more pycnotic and displaced toward the periphery. The cytoplasm is filled with electron transparent vacuoles and vesicles, and dense masses as well as some spiral bodies are seen. The mitochondria retain their shape. Dense particles are seen, which are possibly of viral nature. (c) Final stage. The nucleus is contracted to a narrow strip close to the cellular membrane or is completely destroyed. The cytoplasm shows no apparent changes. Crystals are frequently observed in cells infected with ECHO viruses 6 and 19, consisting of dense particles with an average diameter of 14.4 mµ ranging from approximately 13.2 to 15.6 mµ for ECHO virus 6, and 14.5 mµ ranging from approximately 12.5 to 16.5 mµ for ECHO virus 19. These particles are clustered in hexagonal packages forming angles of 75° and 105°. The particles in most crystals are arranged in rows separated by a constant distance, the latter varying from one crystal to another and being approximately 1.5 and 2.5 times the distance between particles. Other particles were observed which, however, are not considered to be of viral nature.     

The Ultrastructure of Mouse Lung: The Alveolar Macrophage

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1½ hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small (~6 mµ) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.     

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : IV. VIRUSES OF THE RI-APC GROUP

Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mµ in diameter and had a center to center spacing when close packed of about 65 mµ. Many of the less dense particles exhibited an internal body averaging 24 mµ in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body—centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive.     

A cytochemical study on the pancreas of the guinea pig. II. Functional variations in the enzymatic activity of microsomes

Microsomes were isolated from the pancreas of starved and fed guinea pigs. In the first case, the gland was removed from animals starved for 48 hours; in the second, the pancreas was excised 1 hour after the beginning of a meal that ended a fast of 48 hours. These are referred to below as fed animals. In both cases the tissue was homogenized in 0.88 M sucrose and the microsomes obtained by centrifuging the mitochondrial supernatant at 105,000 g for 60 minutes. In starved animals the content of the endoplasmic reticulum of the exocrine cells and the content of the microsomes were found to be of low or moderate density. In fed guinea pigs the cavities of the reticulum frequently contained dense intracisternal granules and the microsomes were distinguished by a content of high density sometimes in the form of recognizable intracisternal granules. In starved animals, the microsomes were found to account for 5 to 20 per cent of the trypsin-activatable proteolytic activity and ribonuclease activity of the whole cell, whereas in fed animals they contained uniformly almost 30 per cent of these activities. In fed animals the dense, cohesive content of the microsomes (intracisternal granules) could be isolated by breaking up the microsomes with dilute (0.1 per cent) deoxycholate solutions and separating microsomal subfractions by differential centrifugation. The specific enzymatic activities of a heavy microsomal subfraction rich in intracisternal granules were almost equal to those of isolated purified zymogen granules. The ribonucleoprotein particles attached to the microsomal membranes could be isolated by the same technique and found also to exhibit some of the same enzymatic activities. Corresponding subfractions isolated from the microsomes of starved animals were considerably less active. The relevance of these findings for the synthesis and intracellular transport of protein in the exocrine cell of the pancreas is discussed.     

NEW CYTOPLASMIC COMPONENTS IN ARTERIAL ENDOTHELIA

A hitherto unknown rod-shaped cytoplasmic component which consists of a bundle of fine tubules, enveloped by a tightly fitted membrane, was regularly found in endothelial cells of small arteries in various organs in rat and man. It is about 0.1 µ thick, measures up to 3 µ in length, and contains several small tubules, ~150 A thick, embedded in a dense matrix, and disposed parallel to the long axis of the rod. In some of these cells, the cisternae of the endoplasmic reticulum are greatly distended by the accumulation of a dense, finely granular material. The nature and significance of these cytoplasmic components are yet unknown.     

AN ELECTRON MICROSCOPE STUDY OF THE CYTOLOGY OF THE PROTOZOAN EUPLOTES PATELLA

1. Structurally the "sensory bristles" in Euplotes patella are typical cilia, but no ciliary rootlets connect their bases. 2. The "neuromotor fibrils" are composed of filaments 21 mµ in diameter. At the point of junction of the filaments with the peripheral ciliary fibrils a granular structure 65 to 90 mµ in diameter is seen which has dense central and peripheral zones separated by a less dense layer. Information on the interconnection of organelles is expanded. 3. A system of subpellicular fibrils is described. The external fibrillar system described by others could not be found. 4. The motorium is shown to be a mass of intertwining rootlet filaments. 5. The micronucleus is shown to have a spongy, dense material in a less dense material, all of which is surrounded by a double-layered membrane. 6. The double-layered macronuclear membrane contains annuli whose outside diameter is 70 mµ; the macronuclear bodies are sometimes closely applied to the membrane. In the macronuclear reorganization bands, the solution plane is a fine network, while the reconstruction plane is devoid of structure at the level of resolution observed. 7. The mitochondria are composed of tubules, only occasionally oriented, usually embedded in a surrounding material of lower density. 8. Microbodies whose diameters are 250 to 350 mµ are frequently observed in close association with mitochondrial surfaces. 9. The food vacuoles, contractile vacuoles, and ciliary vacuoles are bounded by single-layered membranes. In the food vacuoles, the bacteria are surrounded by membranes individually or in small groups. 10. Cytoplasmic rods localized in the oral region, and cytoplasmic granules dispersed at random, are described. No typical ergastoplasm, endoplasmic reticulum, or Golgi material was observed.     

MATURATION OF RAT MAST CELLS : An Electron Microscope Study

Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.     

ELECTRON MICROSCOPE OBSERVATIONS ON INTRACELLULAR VIRUS-LIKE PARTICLES ASSOCIATED WITH THE CELLS OF THE LUCKÉ RENAL ADENOCARCINOMA

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mµ) having a thick capsule and a dense inner body (35 to 40 mµ) that is eccentrically placed within the central cavity (70 to 80 mµ). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.     

USE OF SERIAL SECTIONS TO DELINEATE THE STRUCTURE OF PORTHETRIA DISPAR VIRUS IN THE ELECTRON MICROSCOPE

Consecutive serial sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus revealed bundles of viral rods scattered and oriented at random within the polyhedral body. Each bundle was entirely surrounded by a dense, sharply defined membrane. The rods measured 18 to 22 mµ in diameter and averaged 280 mµ in length. No spherical viral particles were encountered. The effects of variable compression and periodic distortion of the sections on the appearance of the virus are discussed.     

The Fine Structure of the Rod-Bipolar Cell Synapse in the Retina of the Albino Rat

The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mµ in thickness and 400 mµ in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mµ in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.     

A METHOD FOR DETERMINING TOTAL PROTEIN OF ISOLATED CELLULAR ELEMENTS AND CORRESPONDING TRITIUM RADIOACTIVITY

A method is described for the microanalysis of protein, obtained from isolated tissue elements, in the range of 500 µµg-500 mµg. The method entails solubilization of cellular protein with phosphoric acid and heat after extraction of acid-soluble compounds, lipids, and RNA. A procedure for the extraction and recovery of cellular RNA by the use of 40% trichloroacetic acid is presented. The solubilized protein, in the form of a microdroplet, is photomicrographed with monochromatic light at 230 mµ. Total density in the microdroplet is determined from calibrated photographic plates by microdensitometry, and is converted to protein mass by using an experimentally determined average specific absorbance value. A solubilized protein labeled with tritium can be recovered after photomicrography, combusted, and reduced to generate tritiated gas for high-efficiency tritium radiometry. Total protein was analyzed in (a) nerve cells of three different sizes from Deiters' nucleus of the rabbit; and the whole rod cell and rod cell nucleus of the rabbit retina.     

ISOLATION OF RAT PITUITARY GRANULES AND THE STUDY OF THEIR BIOCHEMICAL PROPERTIES AND HORMONAL ACTIVITIES

A simple and rapid method is described for the isolation of the acidophilic and basophilic granules from the anterior pituitary gland of the rat. The method involves chromatography of pituitary particulates on columns of No. 545 Celite equilibrated and developed with 0.25 M sucrose. Mitochondria are retained quantitatively on the column. The granules and microsomes which are not retained on the Celite are further fractionated on a discontinuous density sucrose gradient and by differential centrifugation. Essentially homogeneous populations of acidophilic and basophilic granules were obtained as indicated by 1) extensive electron microscopic studies, 2) enzymatic determinations, and 3) fatty acid and RNA analyses on the granule pellets. Microfiltration studies indicated that the acidophilic granules were smaller than 450 mµ, but greater than 300 mµ in diameter. They were found, unlike the basophilic granules, to be partially stable to extraction with water, but were unstable on incubation in 1 mM EDTA at 37°. Magnesium ions were not detected in the granules. The acidophilic and basophilic granules contained, respectively, 5 and 8 per cent of the protein present in the whole homogenate. Extensive hormone studies showed that growth and lactogenic hormones were associated with the acidophilic granules, while thyroid-stimulating hormone and gonadotropin were associated with the basophilic granules. ACTH was not present in significant amounts in either of the granule fractions, but was localized in a particulate fraction which contained microsomes and small granules. The association of the pituitary hormones with specific granules and cell types is discussed.     

Particle Deposition in a Child Respiratory Tract Model: In Vivo Regional Deposition of Fine and Ultrafine Aerosols in Baboons

To relate exposure to adverse health effects, it is necessary to know where particles in the submicron range deposit in the respiratory tract. The possibly higher vulnerability of children requires specific inhalation studies. However, radio-aerosol deposition experiments involving children are rare because of ethical restrictions related to radiation exposure. Thus, an in vivo study was conducted using three baboons as a child respiratory tract model to assess regional deposition patterns (thoracic region vs. extrathoracic region) of radioactive polydisperse aerosols ([d16–d84], equal to [0.15 µm–0.5 µm], [0.25 µm–1 µm], or [1 µm–9 µm]). Results clearly demonstrated that aerosol deposition within the thoracic region and the extrathoraic region varied substantially according to particle size. High deposition in the extrathoracic region was observed for the [1 µm–9 µm] aerosol (72%±17%). The [0.15 µm–0.5 µm] aerosol was associated almost exclusively with thoracic region deposition (84%±4%). Airborne particles in the range of [0.25 µm–1 µm] showed an intermediate deposition pattern, with 49%±8% in the extrathoracic region and 51%±8% in the thoracic region. Finally, comparison of baboon and human inhalation experiments for the [1 µm–9 µm] aerosol showed similar regional deposition, leading to the conclusion that regional deposition is species-independent for this airborne particle sizes.     

Presence, induction and possible role of glucose 6-phosphatase in mammalian pancreatic islets

1. Pancreatic islets from several mammalian species were investigated for hydrolytic activity towards glucose 6-phosphate. Both the total phosphatase activity towards this substrate and the proportion cleaving glucose 6-phosphate in preference to β-glycerophosphate varied widely between species. In pancreatic-islet homogenates prepared from mice and guinea pigs there was a higher rate of liberation of P_i at pH6·7 from glucose 6-phosphate than from β-glycerophosphate. In these two species cortisone treatment enhanced the enzyme activity towards glucose 6-phosphate but not that towards β-glycerophosphate. Simultaneous injections of ethionine or puromycin blocked this stimulating effect of cortisone. 2. With whole homogenates of mouse pancreatic islets, inverse plots of the relationship between glucose 6-phosphate concentration and enzyme activity suggested the simultaneous action of two enzymes with different K_m values. After fractionation of islets from obese–hyperglycaemic mice by differential centrifugation, one of these enzymes could be shown to be localized in the microsome fraction. It had K_m for glucose 6-phosphate about 0·5mm and optimum pH6·7. It split glucose 6-phosphate in preference to β-glycerophosphate, glucose 1-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate. Incubation of the microsomes at pH5·0 and 37° for 15min. decreased the enzyme activity by about 80%. Glucose was a potent inhibitor, the type of inhibition being neither strictly competitive nor non-competitive. It is suggested that the results indicate the presence of glucose 6-phosphatase in mammalian endocrine pancreas, and that this enzyme may play a role in the metabolic regulation of release of insulin.     

Protection of Pancreatic β-Cells from Various Stress Conditions Is Mediated by DJ-1

Pancreatic β-cells are vulnerable to multiple stresses, leading to dysfunction and apoptotic death. Deterioration in β-cells function and mass is associated with type 2 diabetes. Comparative two-dimensional gel electrophoresis from pancreatic MIN6 cells that were maintained at varying glucose concentrations was carried out. An induced expression of a protein spot, detected in MIN6 cells experiencing high glucose concentration, was identified by mass spectrometry as the oxidized form of DJ-1. DJ-1 (park7) is a multifunctional protein implicated in familial Parkinsonism and neuroprotection in response to oxidative damage. The DJ-1 protein and its oxidized form were also induced following exposure to oxidative and endoplasmic reticulum stress in MIN6 and βTC-6 cells and also in mouse pancreatic islets. Suppression of DJ-1 levels by small interfering RNA led to an accelerated cell death, whereas an increase in DJ-1 levels by adenovirus-based infection attenuated cell death induced by H₂O₂ and thapsigargin in β-cell lines and mouse pancreatic islets. Furthermore, DJ-1 improved regulated insulin secretion under basal as well as oxidative and endoplasmic reticulum stress conditions in a dose-dependent manner. We identified TFII-I (Gtf2i) as DJ-1 partner in the cytosol, whereas the binding of TFII-I to DJ-1 prevented TFII-I translocation to the nucleus. The outcome was attenuation of the stress response. Our results suggest that DJ-1 together with TFII-I operate in concert to cope with various insults and to sustain pancreatic β-cell function.