Kinetics of porphyrin accumulation in cultured epithelial cells exposed to ALA

• 1.1. The kinetics of porphyrin accumulation in cultured mammalian epithelial cells (CNCM-I-221) during exposure to ALA was investigated.
• 2.2. The total porphyrin synthesized is a function of ALA concentration and the incubation time. The cellular porphyrin content exhibited a saturation pattern, reaching a plateau at about 0.04 fmol porphyrins/cell. A biphasic time-dependent increase in the total porphyrin synthesized was observed.
• 3.3. After 3 hr of exposure to ALA the rate of synthesis increased to ahnost twice the initial rate, reaching between 0.02 and 0.05 fmol porphyrins/cell/hr depending on serum concentration in the medium.
• 4.4. Two effects of FBS on ALA-stimulated porphyrin accumulation were observed. Greater total porphyrin synthesis was found when incubations were made in 10% FBS compared to those in 1% FBS.
• 5.5. The higher serum concentration also caused a greater release into the medium of the porphyrins generated in the cells with a calculated half-life of 24 min in 10% serum-supplemented medium compared with 62 min in 1% serum.
• 6.6. The results obtained from cell synchronization experiments suggest that there is little obvious cell cycle-dependent variation in the synthesis of porphyrins from ALA.
• 7.7. The small differences in the intracellular porphyrin content that were observed may be attributed to a slight reduction in the rate of loss of porphyrins in G2/M cells.

     

Contrast in adenine uptake by chicken and rabbit erythrocytes in vitro

• 1.1. Relative to rabbit erythrocytes, chicken red blood cells exhibit a much greater capacity to utilize [³H]adenine for nucleotide synthesis in vitro, even at 5°C and in the absence of added inorganic phosphate.
• 2.2. This difference is largely due to a higher concentration of phosphoribosylpyrophosphate and greater activity of adenine phosphoribosyltransferase in the avian cells. lli]3. The capacity of avian erythrocytes for utilization of guanine and hypoxanthine is several fold less than that of adenine.
• 3.4. The data are consistent with lower activity for hypoxanthine/guanine phosphoribosyltransferase than for adenine phosphoribosyltransferase in intact chicken erythrocytes.
• 4.5. The results indicate that reutilization of adenine by chicken erythrocytes may be physiologically significant.

     

Comparative studies of glucose metabolism on mammals' red blood cells

• 1.1. Glucose utilization was measured in human, pig, cow, rabbit, mouse and rat red blood cells. Mean values are variable from species to species and range from 0.27 μmol/hr/ml RBC for pig erythrocytes to 2.85 μmol/hr/ml RBC in mouse red cells.
• 2.2. The amount of glucose metabolized through the pentose phosphate pathway ranges from 2.1 to 7.0% of the total glucose utilized.
• 3.3. Variable recycling values have been obtained for the red blood cells of the species studied but with the exception of mouse (14 nmol/hr/ml RBC) all the other values do not show great differences.
• 4.4. The hexokinase levels of the erythrocytes studied when correlated with the glucose utilization and the pentose phosphate pathway show that this enzyme could play a central role in the regulation of glucose metabolism.

     

Reduction of ferrihemoglobin in erythrocytes of birds and mammals

• 1.1. Species differences exist in ferrihemoglobin reduction rates in bird and mammalian red cells, bird erythrocytes being very active reducers.
• 2.2. Glucose and lactate enhance ferrihemoglobin reduction. In horse and quail red cells β-hydroxybutyrate has this effect as well.
• 3.3. Malate and pyruvate do not enhance ferrihemoglobin reduction.
• 4.4. Plasma addition to red cell suspensions enhances ferrihemoglobin reduction; addition of lactate mimics this effect in all species except the dog.
• 5.5. Incubation conditions are very important for measuring ferrihemoglobin reduction. Especially the presence of bicarbonate ions is essential. In our experiments no inhibition of reduction rates by chloride ions is found.
• 6.6. Mitochondrial NADH production does not play a role in ferrihemoglobin reduction in bird red cells.

     

Comparison of the porphyrin patterns in patients with porphyria cutanea tarda in Czechoslovakia and Denmark

• 1.1. The incidence of porphyria cutanea tarda (PCT) has increased considerably in Denmark during the last decade (Table 1) but is much higher in Czechoslovakia than in Denmark.
• 2.2. We therefore made a detailed study of the urinary porphyrin excretion pattern in 10 cases of PCT from Copenhagen and 10 from Prague.
• 3.3. The results are presented (Table 2). They show no simple pattern.
• 4.4. Comparison with the type subdivision of Doss et al. (Table 3) and the important findings of Piñol Aguade et al. show that such elaborate type division, involving considerable and time-consuming analytical work, belong to research and have limited value in clinical work.

     

Study of the heme catabolism of fish

• 1.1. The composition of bile pigments in the blood and bile of 39 species were studied.
• 2.2. Conjugated bilirubin (trace to 4.62 mg/100 ml) was detected in the serum of most fish, while biliverdin (trace to 2.0 mg/100ml) was detected only in Anguilla Japonica, Thalassoma lunare and Clinocottus analis.
• 3.3. Analysis showed tht there are two types of bile pigments excretion pattern in these fishes. The first pattern excretes bilirubin (most conjugate) predominantly, the other excretes mostly biliverdin with some bilirubin. However, during starvation, the excretion of conjugate bilirubin gradually shifted to unconjugated biliverdin. The rate of shifting varies with species.
• 4.4. Introduction of bilirubin into Anguilla japonica produced an initial excretion of mono-conjugates, followed by di-conjugates. Introduction of biliverdin caused an increased in the excretion of unconjugated biliverdin, but no significant increase of bilirubin in the bile was detected.
• 5.5. A binary excretion pathway of bile pigments in fish is proposed. The evolutionary characteristics of heme catabolism in terrestrial animals with respect to this pathway is discussed.

     

The peculiarities of nitrogen excretion in sturgeons (Acipenser ruthenus) (Pisces,Acipenseridae)—I. the influence of ration size

• 1.1. The nonfaecal nitrogenous excretion rate in starved sterlet fingerlings and fingerlings fed on different rations was investigated. The weight of the fish and temperature of the water was 43 g and 17.5°C, respectively.
• 2.2. In the nonfaecal excrements of starved sterlets the ammonia: urea ratio was substantially lower than in teleosts. This ratio was found to be 1.4:1.
• 3.3. In fed sterlets the urea excretion rate was higher than in starved ones but independent of ration size.
• 4.4. During the day the urea excretion rate in sterlets was constant.
• 5.5. The ammonia excretion rate accelerated 2 hr after feeding and reached its peak duration 6–11 hr after depending on the ration size.
• 6.6. Total ammonia output in the sterlet increased following the increase of ration size up to 8.4% of body wt. Further increases in ration size did not cause the corresponding elevation of ammonia excretion rate.

     

Urea excretion rates and waste nitrogen concentrations during metamorphosis of Xenopus laevis Daudin

• 1.1. Serum urea, ammonia concentrations in the blood and excretion were measured in tadpoles of different stages and juveniles of Xenopus laevis.
• 2.2. The urea excretion rate was determined with the help of injected ¹⁴C-urea.
• 3.3. Urea concentrations are higher during metamorphic climax and at the end of metamorphosis than during prometamorphosis.
• 4.4. Blood ammonia levels remain rather constant throughout metamorphosis.
• 5.5. Coincidentally, the relative amount of urea in the blood increases.
• 6.6. The ¹⁴C-urea excretion rates slow down from very high values (48%/hr) at the beginning of prometamorphosis to low rates (5%/hr) in newly metamorphosed animals.
• 7.7. This means that during metamorphosis not only is the possibility of urea production established. but there is a capacity to retard and store urea to some extent.

     

Phosphate compounds in reptilian and avian red blood cells; developmental changes

• 1.1. Red blood cells were analyzed for ATP, 2,3-diphosphoglycerate and inositol polyphosphate at different stages of development of two birds; pigeon and Western gull, and several reptiles: Pacific Ridley, snapping and Pseudemys turtles; green iguana, yellow rat snake and American alligator. Other species were examined only as juveniles or adults: ostrich, Boa constrictor and the Nile and Moreleti crocodiles. The results were compared with the phosphate compounds found in red cells of adult man, rat and rabbit.
• 2.2. The presence, concentration, and time of occurrence of diphosphoglycerate and inositol polyphosphate, separately or together, during development, provided new biochemical clues to evolutionary relationships.
• 3.3. Remarkably large differences in the concentration of ATP in red cells during the development of an animal, and among different kinds of animals, could not be explained.

     

The taurine content of avian erythrocytes and its role in osmoregulation

• 1.1. The taurine content of erythrocytes from 15 avian species contained levels of taurine in the range of 20–70 mmol/kg of hemoglobin, about 100-fold that of mammalian red blood cells.
• 2.2. This high taurine content did not appear to be related to the nucleation of these cells as nucleated amphibian erythrocytes and human reticulocytes contained low levels.
• 3.3. The erythrocytes lacked cysteine sulfinic acid decarboxylase, a key enzyme in the synthesis of taurine from cysteine, indicating a probable lack of synthetic capabilities.
• 4.4. The cells were able to accumulate labeled taurine against a concentration gradient. This uptake was inhibited by β-alanine and was Na⁺-dependent.
• 5.5. When incubated in hypotonic medium, the cell volume of pigeon erythrocytes rapidly increased and was followed by a much slower return to normal size. The cell volume reduction was accompanied by a slow efflux of taurine into the medium.
• 6.6. These data suggest that taurine plays a role in cell volume maintenance and osmotic regulation in avian erythrocytes.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

Haematology of the australian eastern quoll,Dasyurus viverrinus

• 1.1. A variety of haematological parameters were determined in adult Dasyurus viverrinus.
• 2.2. Haemoglobin and red cell counts were high with a very low mean cell volume.
• 3.3. Basophils are absent but the eosinophils contain small numbers of basophilic granules which may indicate a dual role for this cell.
• 4.4. “Ring Form” leucocytes are present.
• 5.5. Three types of red cell picture could be identified, some animals showing large numbers of spherocytes, spicule cells, and inclusion bodies.
• 6.6. These cells resemble those found in some inherited human haemolytic anaemias but there was no evidence of haemolysis in the animals.
• 7.7. An alkali resistant haemoglobin component is present.

     

Investigation of ribosome inactivating protein-like activity in tissues of cucurbitaceae plants

• 1.1. Extracts of roots, seeds and fruits of seventeen plant species belonging to Family Cucurbitaceae were examined for the ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Out of the 22 tissue extracts examined, 16 were found to inhibit protein synthesis by >90%, three caused 65–85% inhibition and 3 caused <25% inhibition.
• 3.3. In general, there was a close correlation between protein synthesis inhibiting activity and mid-term abortifacient activity of the tissue extracts.
• 4.4. SDS-PAGE of the tissue extracts revealed the presence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000. The data suggest that this band is responsible for the protein synthesis inhibiting and mid-term abortifacient activities.

     

Ribosome inactivating protein-like activity in seeds of diverse Cucurbitaceae plants

• 1.1. Seed extracts of 20 plants species belonging to the family Cucurbitaceae were examined for their ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Eleven extracts were found to inhibit protein synthesis by about or over 90%, seven extracts produced about 80% inhibition, one caused about 70% inhibition and one brought about approx. 40% inhibition, when the extracts were tested at a final concentration of 10 μg per ml.
• 3.3. All of the seed extracts possessed potent mid-term abortifacient activity.
• 4.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the seed extracts disclosed the existence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000 Da. This band probably accounts for the protein synthesis inhibiting and mid-term abortifacient activities.
• 5.5. There was a similarity in the electrophoretograms of seed extracts of plants belonging to the same genus.

     

Effects of membrane-perturbing drugs and noradrenalin on cAMP accumulation and red cell water content in carp (Cyprinus carpio) red cells

• 1.1. Carp red cells were treated with drugs that affect the cell membranes. The water content of the cells and the accumulation of cAMP in the cells were measured in normoxia and in hypoxia using non-stimulated and adrenergically stimulated cells.
• 2.2. WGA, DIDS + CCCP and A23187 increased the water content of nonstimulated normoxic cells.
• 3.3. In hypoxia ouabain and DIDS + CCCP increased the water content but cytochalasin B, NPM, DIDS, CCCP and A23187 + CA²⁺ abolished the hypoxia-induced swelling.
• 4.4. Any membrane perturbation induced some cAMP formation, Sophora and Anquilla lectins being most potent.
• 5.5. Also in adrenergically stimulated cells, membrane perturbation generally increased cAMP formation.
• 6.6. However, cAMP accumulation diminished in cells treated with cytochalasin B, CCCP and DIDS + CCCP.
• 7.7. The adrenergic swelling of carp red cells was reduced in normoxia by DIDS. NPM and CCCP increased the adrenergic swelling in normoxia to hypoxic level.
• 8.8. In hypoxia WGA and Anquilla lectin decreased the swelling.

     

Diversity of native myosin and myosin heavy chain in fish skeletal muscles

• 1.1. Polymorphism of native myosin and myosin heavy chain (MHC) of fish skeletal muscles was analysed by pyrophosphate and SDS-gel electrophoreses.
• 2.2. Depending on the species, three or four myosin isoforms were detected in the white muscle, one or two isoforms in the pure red muscle, and four isomyosins were found in the red muscle composed of red and pink (intermediate) fibres.
• 3.3. It is suggested that all main types of fish muscle fibre (red, intermediate and white) differ in myosin isoform content.
• 4.4. Myosin heavy chain of the red muscle is a distinct protein from that of the white muscle. However, structural differences between these proteins vary among species.

     

Acute hepatic porphyria syndrome with porphobilinogen synthase defect

On the basis of metabolite and enzyme studies a new type of acute hepatic porphyria with porphobilinogen synthase defect and repeated intermittent acute manifestations, abdominal colics, tachycardia and hypertension, and a persistent neurological syndrome was found in two young male patients. The main characteristic features are the following:

• 1.1. High urinary δ-aminolevulinic acid excretion( ⪢ 1 mmol/24hr), slight increase of porphobilinogen (up to 25 μmol/24 hr) and high increase of porphyrins (up to 22 μmol/24 hr) with coproporphyrin dominance.
• 2.2. Normal fecal and liver porphyrins.
• 3.3. Slight increase of erythrocyte protoporphyrin.
• 4.4. Decrease of porphobilinogen synthase activity in erythrocytes in both cases below 1% of healthy and not lead-exposed persons; normal activities of uroporphyrinogen synthase and decarboxylase in erythrocytes.
• 5.5. Low-normal lead concentrations in blood and low-normal lead excretion in urine in both cases; normal lead content in bone.
• 6.6. Normal plasma and urinary amino acids.
• 7.7. Irrelevant hepatological (liver biopsy), general clinical chemical and hematological findings.
• 8.8. Diminished activity of porphobilinogen synthase in nearly all family members of both patients. From these investigations it can be concluded that there is no exogeneous, “toxic” cause of this porphyria. Porphobilinogen synthase in lead poisoning is not diminished to such an extent as demonstrated here; in contrast to lead intoxication, porphobilinogen synthase activity cannot be activated or reactivated by thiols. All clinical and pathobiochemical data point at a new enzymatic type of endogeneous acute hepatic porphyria with intermittent acute manifestations, clinically analogous to so-called acute intermittent porphyria. Porphyrin precursors and porphyrin excretion both reflects the enzymatic defect and the regulatory consequences starting with the induction of δ-aminolevulinic acid synthase.

     

δ-Aminolevulinic acid dehydratase inactivation by uroporphyrin I in light and darkness

• 1.1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined.
• 2.2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed.
• 3.3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin.
• 4.4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein.
• 5.5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

     

Cell surface labeling by iodine monochloride

• 1.1. Human red cell membranes were trace iodinated with [¹²⁵I]ICI and the distribution of label in membrane components examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 2.2. With intact erythrocytes over 84% of the bound iodine was associated with the membrane.
• 3.3. Two membrane components accounted for almost all of the label, band 3 and PAS-1.
• 4.4. Spectrin was not labeled in resealed ghosts.

     

Arginase activity in different fish species and tissues

• 1.1. Arginase activity was measured in different tissues from eight species of fish.
• 2.2. Spur dogfish showed a very high arginase activity compared with the other species analysed.
• 3.3. The activity in teleosts was mainly found in tissues of high metabolic activity (liver, kidney and red muscle).