scholarly journals A Cytochemical Study on the Pancreas of the Guinea Pig

1960 ◽  
Vol 7 (4) ◽  
pp. 619-630 ◽  
Author(s):  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Guinea Pig ◽  
Microsomal Fraction ◽  
Specific Radioactivity ◽  
Early Time ◽  
Acid Extraction ◽  
Time Intervals ◽  
Cytochemical Study ◽  
Cell Compartments ◽  
Exocrine Cell ◽  
Cell Fractions

Chymotrypsinogen synthesis in the exocrine cell of the guinea pig pancreas was studied under the following conditions: Animals fed after a fast of ∼48 hours received ∼1 hour after feeding an intravenous injection of DL-leucine-1-C14. At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animals were used for each time point. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH4)2SO4 fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.

scholarly journals Adenine derivatives as neurohumoral agents in the brain. The quantities liberated on excitation of superfused cerebral tissues

1972 ◽  
Vol 130 (4) ◽  
pp. 975-981 ◽  
Author(s):  
Ian Pull ◽  
Henry McIlwain
Keyword(s):  
Electrical Stimulation ◽  
Guinea Pig ◽  
Adenine Nucleotides ◽  
Specific Radioactivity ◽  
Acid Extraction ◽  
Free Medium ◽  
Hydrolysis Of ◽  
The Brain ◽  
Stimulation Of ◽  
Adenine Derivatives

Adenine nucleotides of guinea-pig neocortical tissues were labelled by incubation with [14C]adenine and excess of adenine was then removed by superfusion with precursor-free medium. Adenine derivatives released from the tissue during continued superfusion, including a period of electrical stimulation of the tissue, were collected by adsorption and examined after elution and concentration. The stimulation greatly increased the 14C output, and material collected during and just after stimulation had a u.v. spectrum which indicated adenosine to be a major component. The additional presence of inosine and hypoxanthine was shown by chromatography and adenosine was identified also by using adenosine deaminase. Total adenine derivatives released from the tissue during a 10min period of stimulation were obtained as hypoxanthine, after deamination and hydrolysis of adenosine and inosine, and amounted to 159nmol/g of tissue. This corresponded to the release of approx. 7pmol/g of tissue per applied stimulus. The hypoxanthine sample derived from superfusate hypoxanthine, inosine and adenosine was of similar specific radioactivity to the sample of inosine separated chromatographically, and each was of higher specific radioactivity than the adenine nucleotides obtained by cold-acid extraction of the tissue.

scholarly journals A Cytochemical Study on the Pancreas of the Guinea Pig

1958 ◽  
Vol 4 (2) ◽  
pp. 203-218 ◽  
Author(s):  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Electron Microscopy ◽  
Guinea Pig ◽  
Pancreatic Tissue ◽  
Supernatant Fraction ◽  
Differential Centrifugation ◽  
Zymogen Granules ◽  
Cytochemical Study ◽  
Specific Activities ◽  
Main Components ◽  
Cell Fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.

scholarly journals A Cytochemical Study on the Pancreas of the Guinea Pig

1958 ◽  
Vol 4 (5) ◽  
pp. 557-566 ◽  
Author(s):  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Pancreatic Cell ◽  
Cytochemical Study ◽  
Mitochondrial Fractions ◽  
Liver And Pancreas ◽  
Intracardiac Injection ◽  
Cell Fractions

DL-leucine-1-C14 was administered by intracardiac injection to guinea pigs and its in vivo incorporation into the proteins of various pancreatic cell fractions followed over a period of 2 hours. The pancreas was homogenized in 0.88 M sucrose and fractionated by differential centrifugation to give nuclear, zymogen, mitochondrial, microsomal, postmicrosomal, and final supernatant fractions. The proteins of these fractions, obtained by precipitation with trichloroacetic acid followed by washing, were counted. The proteins of the microsomal fraction showed the highest early specific activity and were followed by those of the zymogen and mitochondrial fractions. The microsomal fraction was broken up into two subfractions: one consisting of detached RNP particles, the other representing mainly the microsomal content and membranes. The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancreas of fasted animals. A tentative cytological picture of protein synthesis and transport based on these findings is presented.

scholarly journals The fate of 14C in glucose 6-phosphate synthesized from [1-14C]Ribose 5-phosphate by enzymes of rat liver

1978 ◽  
Vol 176 (1) ◽  
pp. 241-256 ◽  
Author(s):  
J F Williams ◽  
M G Clark ◽  
P F Blackmore
Keyword(s):  
Rat Liver ◽  
Pentose Phosphate Pathway ◽  
Specific Radioactivity ◽  
Early Time ◽  
Pentose Phosphate ◽  
Enzyme Extract ◽  
Reaction Sequence ◽  
Time Intervals ◽  
A Value ◽  
Initial Substrate

1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1–30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3–17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the ‘defined’ pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation.

INCORPORATION OF IODIDE INTO ORGANIC COMPOUNDS IN THE GUINEA PIG THYROID

1963 ◽  
Vol 43 (1) ◽  
pp. 110-118 ◽  
Author(s):  
R. Ekholm ◽  
T. Zelander ◽  
P.-S. Agrell
Keyword(s):  
Guinea Pig ◽  
Protein Binding ◽  
Organic Compounds ◽  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Thyroid Tissue ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hydrolysis Of

ABSTRACT Guinea pigs, kept on a iodine-sufficient diet, were injected with Na131I and the thyroids excised from 45 seconds to 5 days later. The thyroid tissue was homogenized and separated into a combined nuclear-mitochondrial-microsomal fraction and a supernatant fraction by centrifugation at 140 000 g for one hour. Protein bound 131iodine (PB131I) and free 131iodide were determined in the fractions and the PB131I was analysed for monoiodotyrosine (MIT), diiodotyrosine (DIT) and thyroxine after hydrolysis of PB131I. As early as only 20 minutes after the Na131I-injection almost 100% of the particulate fraction 131I was protein bound. In the supernatant fraction the protein binding was somewhat less rapid and PB131I values above 90% of total supernatant 131I were not found until 3 hours after the injection. In all experiments the total amount of PB131I was higher in the supernatant than in the corresponding particulate fraction. The ratio between supernatant PB131I and pellet PB131I was lower in experiments up to 3 minutes and from 2 to 5 days than in experiments of 6 minutes to 20 hours. Hydrolysis of PB131I yielded, even in the shortest experiments, both MIT and DIT. The DIT/MIT ratio was lower in the experiments up to 2 hours than in those of 3 hours and over.

scholarly journals Evidence for the presence of stem cell-like progenitor cells in human adult pancreas

2007 ◽  
Vol 195 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Min Zhao ◽  
Stephanie A Amiel ◽  
Michael R Christie ◽  
Paolo Muiesan ◽  
Parthi Srinivasan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Adult Life ◽  
Stem Cell Markers ◽  
Human Pancreas ◽  
Β Cells ◽  
Adult Human ◽  
Pancreatic Cells ◽  
Exocrine Cell ◽  
Cell Fractions ◽  
Unique Cell

The origin of cells replacing ageing β-cells in adult life is unknown. This study assessed the expression of classic stem cell markers: Oct4, Sox2 and CD34 in islet-enriched fractions versus exocrine cell-enriched fractions from 25 adult human pancreases following human islet isolation. Expression of Oct4, Sox2 and CD34 mRNAs was found in all cell samples, with no significant differences between endocrine and exocrine cell fractions. Immunohistochemical staining for Oct4, Sox2, CD133, CD34, CK19, insulin and nestin on human pancreas sections showed that the majority of Oct4+ve cells were found in the walls of small ducts. Similar localisations were observed for Sox2+ve cells. The majority of Sox2+ve cells were found to co-express Oct4 proteins, but not vice versa. Cells positive for Oct4 and Sox2 appeared to be a unique cell population in the adult human pancreases without co-expression for CK19, CD34, CD133, insulin and nestin proteins. The numbers of Oct4+ve and Sox2+ve cells varied among donors and were ∼1–200 and 1–30 per 100 000 pancreatic cells respectively.

Studies on the effects of magnesium ion and propranolol on iris muscle phosphatidate phosphohydrolase

1984 ◽  
Vol 62 (2-3) ◽  
pp. 170-177 ◽  
Author(s):  
Ata A. Abdel-Latif ◽  
Jack P. Smith
Keyword(s):  
Smooth Muscle ◽  
Divalent Cation ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Divalent Cations ◽  
Dependent Manner ◽  
Soluble Enzyme ◽  
Microsomal Enzyme ◽  
Time Intervals ◽  
Phosphatidate Phosphohydrolase

The properties, subcellular distribution, and the effects of Mg2+ and propranolol on phosphatidate phosphohydrolase (EC 3.1.3.4) from rabbit iris smooth muscle have been investigated. The particulate and soluble (0–30% (NH4)2SO4 fraction) enzymes were assayed using aqueous phosphatidate dispersions and membrane-bound phosphatidate as substrates, respectively. When measured with aqueous substrate, activity was detected in both the particulate and soluble fractions, with the highest relative specific activity found in the microsomal fraction. Maximum dephosphorylation by the microsomal enzyme was about 1100 nmol of inorganic phosphate released/h per milligram protein and occurred at pH 7.0–7.5. In general Mg2+ inhibited the phosphohydrolase activity of the microsomal fraction and stimulated that of the soluble fraction, and the effects of the divalent cation on both of these activities were reversed by propranolol. The microsomal enzyme was slightly stimulated by deoxycholate and inhibited by the divalent cations Mg2+, Ca2+, and Mn2+ at concentrations > 0.25 mM. In contrast, the soluble enzyme was stimulated by Mg2+. Inhibition of the microsomal enzyme by Mg2+ (0.5 mM) was reversed by both EDTA, which also stimulated at higher concentrations (1 mM), and propranolol (0.1–0.2 mM). The inhibitory effect of Ca2+ on the enzyme was not reversed by propranolol. In the absence of Mg2+, the microsomal enzyme was inhibited by propranolol in a dose-dependent manner, and both in the absence and presence of the divalent cation the soluble enzyme was inhibited by the drug in a similar manner. These data suggest that the cationic moiety of propranolol may act by competing at the Mg2+-binding sites. Addition of propranolol (0.2 mM) to iris muscle prelabelled with [14C]arachidonic acid increased accumulation of [14C]phosphatidic acid at all time intervals (2.5–90 min) and brought about a corresponding initial decrease in the formation of [14C]diacylglycerol at short time intervals (2.5 min), thus implicating the phosphohydrolase as a possible site of action of the drug on glycerolipid metabolism in this tissue. In addition to reporting on the characteristics and distribution of phosphatidate phosphohydrolase in the iris smooth muscle, the data presented add further support to our hypothesis that propranolol redirects glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in their biosynthesis.

scholarly journals Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1970 ◽  
Vol 119 (3) ◽  
pp. 575-585 ◽  
Author(s):  
M. J. Barnes ◽  
B. J. Constable ◽  
L. F. Morton ◽  
E. Kodicek
Keyword(s):  
Molecular Weight ◽  
Ascorbic Acid ◽  
Guinea Pigs ◽  
Specific Radioactivity ◽  
Rapid Decline ◽  
Time Intervals ◽  
Skin Collagen ◽  
Labelled Compound ◽  
Degree Of Degradation

1. After the administration of l-[G-3H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.

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