Cell line A549 as a model of the type II pneumocyte. Phospholipid biosynthesis from native and organometallic precursors. 1. A549 is a continuous cell line derived from a human pulmonary adenocarcinoma. To evaluate the suitability of this cell line as a model of the type II pneumocyte, the morphology and the composition and biosynthesis of phosphatidylcholine was examined under control culture conditions and during fatty acid supplementation with palmitate. A number of the ultrastructural characteristics of A549 cells were similar to the in situ type II pneumocyte and were unchanged by fatty acid supplementation. The phospholipid composition of the cell line was similar to that of primary isolates of type II cells in total phosphatidylcholine, disaturated phosphatidylcholine, and palmitate and saturated fatty acid. Phospholipid biosynthetic results were also consistent with those reported for isolated type II cell models. These included: (i) the pattern of incorporation of choline, palmitate and acetate into phosphatidylcholines; (ii) the effect of palmitate supplementation, which resulted in stimulation of the rate of phosphatidylcholine biosynthesis and in increased percentage of labeled precursor in disaturated phosphatidylcholine; and (iii) the preferential synthesis from labeled choline and palmitate of a highly disaturated phosphatidylcholine in short-term incubations. 2. The incorporation of an organometallic palmitate analog, 12,12-dimethyl-12-stannahexadecanoate, into A549 cell lipids was examined and compared to that of palmitate. These date demonstrate for the first time the incorporation of an organometallic substrate into the phospholipids of a mammalian cell line. This analog substitutes selectively for the native fatty acid at a rate similar to that of the native fatty acid with no cytotoxic effects. The organotin probe, coupled with spectroscopic detection and electron microscopy, may be useful for examining ultrastructural aspects of phospholipid synthesis, translocation and assembly.


Isolation and identification of polyprenols from bovine thyroid gland. The presence of polyprenols in bovine thyroid was demonstrated. After preparative isolation, the structure was elucidated by chemical and spectroscopic techniques. The main polyprenol homologue has a molecular weight of 1380 corresponding to the presence of 20 isoprene units. From NMR studies it appears that 18 units have the cis configuration and that the 2 others are trans isoprene units. The dolichol content amounts to 0.2 mg/g wet weight. About 5% was found in the esterified form.


The lipids of the Golgi apparatus subfractions from rat liver. Golgi apparatus were isolated from untreated rat liver and separated into three fractions. One consisted mainly of vesicles, a second of tubular particles (dictyosomes) and the third was a mixed fraction. Large differences between these fractions could be seen in the electron microscope and by enzyme analysis. The total lipid content of the vesicles was 3.5-times greater than that of the dictyosomes and the neutral lipid value was 7-times greater. The ratio of phospholipids to protein was approximately the same in the three fractions. However, the phospholipid patterns differed between the vesicle and dictyosome fractions.


Regulation of body cholesterol pools. Influence of cholesterol input and excretion in an animal model. Biliary cholesterol excretion closely parallels lecithin excretion in the rat and may be increased by feeding an excess of choline and decreased by choline deficiency. To determine the relative influence of cholesterol input and excretion on whole body cholesterol metabolism, we have measured by compartmental analysis rates of cholesterol transport and pool sizes when both input and choline-mediated biliary excretion were increased and diminished. In choline-deficient animals with impaired excretion, plasma cholesterol was reduced. However, in deficient animals more cholesterol was transported into the slowly exchanging pool to increase pool size, and, when input was increased (by addition of cholesterol to diet), the slowly exchanging pool was even more markedly enlarged. In contrast, when excess choline was fed, plasma cholesterol was increased but excretion so exceeded transport into the slowly exchanging pool that pool size was actually reduced. Furthermore, in choline-fed animals with unimpaired excretion, addition of cholesterol to the diet to increase input did not result in pool expansion. Thus, in this model, cholesterol excretion and tissue deposition were reciprocally related, and, regardless of plasma cholesterol concentration and cholesterol input, stores were found to increase only when biliary excretion was impaired.


Stimulation of cholesterol esterification in hepatic microsomes by lipoproteins from normal and hypercholesterolemic rabbit serum. Incubation of plasma lipoproteins with rabbit hepatic microsomes enriched the microsomes with free cholesterol and stimulated cholesterol esterification. The rate of cholesterol esterification correlated well (r = 0.96) with the concentration of microsomal free cholesterol. Lipoproteins from normal and hypercholesterolemic serum varied in their propensity to stimulate cholesterol esterification. Among the normal lipoproteins, low density lipoproteins was more stimulatory than either high density lipoproteins or intermediate density lipoproteins. However, the intermediate density lipoproteins fraction from hypercholesterolemic serum was consistently more stimulatory than any of the normal lipoproteins. The augmentation of cholesterol content, when microsomes were exposed to mixed hyperlipidemic lipoproteins, was proportionately much greater than augementation of phospholipid or protein concentration.


The kinetics of binding of serum lipoproteins by immobilized heparin. A model arterial system of heparin immobilized on an agarose gel was used to study the amount and kinetics of binding of porcine serum lipoproteins to heparin. Binding occurred to lipoproteins in the density range 1.006 less than d less than 1.062, but there was no binding with high density lipoprotein. A theoretical model of the kinetic experiments was formulated and used to demonstrate that the rate of the binding reaction could be considered instantaneous relative to the rate of transport of lipoproteins. Extrapolation of these results to arterial levels of glycosaminoglycans and lipoprotein indicate that complexes of lipoprotein and the glycosaminoglycans could account for much of the cholesterol entrapment in atherosclerotic lesions.


The effect of cerulenin and exogenous fatty acids on triacylglycerol accumulation in an inositol-deficient yeast, Saccharomyces carlsbergensis. Cerulenin inhibits fatty acid synthesis in yeast; supplementation with exogenous fatty acids is required to maintain cell growth. In the presence of cerulenin and exogenous fatty acids inositol-deficient cells accumulate triacylglycerols to almost the same extent as normally grown deficient cells, indicating that increased fatty acid synthesis is not primarily responsible for triacylglycerol accumulation.


Stimulation of thiamine diphosphatase activity by ascorbic acid in rat brain microsomes. The effect of ascorbic acid on microsomal thiamine diphosphatase activity in rat brain was examined. Ascorbic acid at 0.02--0.1 mM increased the thiamine diphosphatase activity by 20--600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, alpha,alpha'-dipyridyl, o-phenanthroline) and an antioxidant (N,N'-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02--0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.


Proteolytic enzymes in normal and transformed cells. Virally transformed cells show an increased production of proteolytic enzymes. These might be involved in transformation-dependent alterations of cell surface glycoproteins. The possibility arises that some of these proteases might be membrane-bound. To investigate this possibility, we have undertaken a comparative study of the reactivity of intact normal and transformed cells with the tritium labelled protease inhibitor diisopropylfluorophosphate, in parallel with fibrinolytic assays. Using these two approaches in concert, it was possible to identify and localize in the transformed cells several proteases which were present in the particulate cell fraction and were probably membrane bound. In particular, a diisopropylfluorophosphate-reactive polypeptide of 62,000 was increased 5--8-fold on transformation. It comigrated with a fibrinolytic activity. Other particle-bound activities were also detected. While diisopropylfluorophosphate-labelling can be useful for detecting proteases inside cells, it does not appear to be specific for surface proteases.


Effect of lectins on endocytosis and secretion of lysosomal enzymes by cultured fibroblasts. 1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of alpha-N-acetylglucosaminidase and increased extracellular accumulation of beta-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl alpha-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited alpha-N-acetylglucosaminidase endocytosis, but had no effect on extracellular beta-N-acetylglucosaminidase accumulation. 4. Concanavalin A-alpha-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.


Characterization of the sulfated glycosaminoglycan on the surface and in the storage granules of rabbit platelets. Rabbit platelets were labeled in vivo with 35S for characterization of platelet sulfated glycosaminoglycan. When rabbit platelets were aggregated by ADP, sulfated proteoglycan was lost from the platelet surface although no release of granule contents occurred. The sulfated proteoglycan contained in the granules of platelets pretreated with ADP was subsequently released by treatment with thrombin. The 35S-labeled proteoglycan from both sources was isolated by gel filtration and the glycosaminoglycan portion of the proteoglycan was characterized as chondroitin 4-sulfate by examining the products of digestion with hyaluronidase, chondroitinase AC and ABC, and chondro-4- and 6-sulfatases; by identification of the hexosamine as N-acetylgalactosamine; by determination of a 1 : 1 : 1 molar ratio of N-acetylgalactosamine, uronic acid and inorganic sulfate; and by cetylpyridinium chloride cellulose chromatography. In these studies, the use of 35S-labeled proteoglycan made possible detection and quantification of much smaller amounts of material than would be possible with unlabeled material. Chondroitin 4-sulfate was the only sulfated glycosaminoglycan identified in the proteoglycan lost from the platelet surface during ADP-induced aggregation and in the proteoglycan released from the granules when the platelets were exposed to thrombin.


Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis. Collagen metabolism was studied in degenerative articular cartilage of dogs with spontaneous, early onset osteoarthritis. A fraction of collagen which represented about 1.5% of the total was extracted from cartilage samples with dilute phosphate buffer (pH 7.4) containing 0.2% sodium dodecyl sulfate. Agarose gel filtration in the presence of sodium dodecyl sulfate revealed that extracts of degenerative cartilage had about 24% procollagen whereas extracts of normal samples had only 3%. The isolated procollagen fraction was rechromatographed on agarose columns in the presence of mercaptoethanol. This resulted in the identification of a collagen species which migrated between marker beta and alpha collagen chains. The molecular weight of this collagen was estimated to be 150,000. Based on incorporation of [14C]proline, its ratio of hydroxy[14C]proline to total 14C was 0.32. Procollagen was not found after limited pepsin digestion (pH 3, 4 degrees C, 16 h) of degenerative cartilage samples. Since the total collagen content (microgram hydroxyproline/mg cartilage), hydroxy-[14C]proline/mg cartilage, specific radioactivity of hydroxyproline in the extractable collagen fraction were similar for normal and degenerative cartilage we propose that procollagen accumulated in the degenerative cartilage due to a partial defect in conversion of procollagen to collagen.


Characteristics of the vitamin K-dependent carboxylating system in human placenta. Gamma-carboxyglutamic acid, formed during the post-translational vitamin K-dependent carboxylation of glutamic acid residues in polypeptides has been identified not only in coagulation factors II (prothrombin),, VII, IX and X [1--4], but also in several other plasma proteins [3,5,6] and in protein of bone [7,8] and kidney [9]. In rat liver, carboxylation is mediated through an enzyme system located in the microsomal membrane [10]. The enzyme system requires CO2, O2 and the reduced (hydroquinone) form of the vitamin, as well as a suitable substrate [10,11]. Rat liver microsomes also convert vitamin K1 (phylloquinone) to its stable 2,3-epoxide [12]. Several studies suggest a link between carboxylation and the formation of the epoxide [12--14]. In one of these [14], a survey of rat tissues for vitamin K1 epoxidation revealed that, in addition to liver, this activity was also possessed by kidney, bone, spleen and placenta. In preliminary experiments, vitamin K-dependent carboxylating systems have been found in rat and chick kidney [9], in chick bone [15] and in rat spleen and placenta (unpublished observations). In this communication, we describe some of the basic characteristics of the vitamin K-dependent carboxylating system as found in human placental microsomes.


Amylase release from rat parotid glands. II. Calcium kinetics. The kinetics of 45Ca2+ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM 45Ca2+ medium increased the total tissue 45calcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on 45Ca2+ kinetics, or total 45calcium content. Carbamylcholine increased the initial rate of 45Ca2+ uptake, but had no effect on total uptake. Elevation of the extracellular Ca2+ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40--50 min to reach the maximal response. The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca2+ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.


In vitro metabolism of selenite in sheep blood: factors controlling the distribution of selenium and the labelling of plasma protein. Some factors controlling the distribution of Na275SeO3 in sheep blood were studied in vitro. After centrifuging Na275SeO3-incubated blood most of the radioactivity was found in the plasma. The labelling of plasma protein by 75Se was dependent on the presence of erythrocytes. The degree of labelling of plasma protein increased with erythrocyte concentration. When phosphate-buffered saline-washed erythrocytes were suspended in phosphate-buffered saline and incubated with Na275SeO3 the majority of the 75Se was detected in the erythrocytes. On incubating these labelled erythrocytes with unlabelled plasma there was a transfer of radioactivity to the plasma. The calculated activation energy for the labelling of plasma was 107.52 kJ/mol. Albumin was shown not to be a principal acceptor of 75Se from the erythrocytes by ammonium sulphate precipitation of radioactive plasma. Addition of Na2SeO3 to the labelled blood resulted in the transfer of 75Se from plasma to the erythrocytes. Radioactive plasma incubated at 37 degrees C was thermolabile with respect to its 75Se content whereas in whole blood the degree of 75Se binding to plasma protein did not vary suggesting that a recycling of selenium was occurring in blood. From the results presented an in vitro model of selenium metabolism in blood is postulated.


Glucocorticoid receptors in WI-38 fibroblasts: characterization and changes with population doubling in culture. A high-affinity dexamethasone binding macromolecule was identified in WI-38 human fetal lung fibroblasts. High specificity of binding for glucocorticoids was shown by competition studies in which binding of dexamethasone was inhibited by cortisol and corticosterone but not by testosterone or 17 beta-estradiol. WI-38 cells exposed to [3H]dexamethasone at 30 degrees C were able to transfer the 3H-labeled steroid-receptor complex to the nuclear materal. A reduction of 30--50% was observed in the number of [3H]dexamethasone-receptor binding sites per cell as well as in the nuclear fraction of the cells as a function of age (passage levels 27 and 54). However, in the same cells no significant changes in affinity of receptor for [3H]dexamethasone as a function of the two passage levels were detected.


Corticosterone binding in AtT-20 pituitary tumor cell cytosol: evidence for one class of binding site for both natural and synthetic glucocorticoids. The AtT-20 mouse pituitary cell is an established, cloned cell line which produced adrenocorticotrophic hormone in a glucocorticoid-suppressible manner. A receptor for glucocorticoids was identified in cytosol prepared from these cells using the natural mouse glucocorticoid, corticosterone, as the labeled ligand. The question of whether this binding component is identical to the one detectable using labeled triamcinolone acetonide was addressed by comparing their physicochemical characteristics and by detailed studied of binding specificity using both ligands. The corticosterone and triamcinolone acetonide binding components behaved similarly on sucrose density gradient analysis and DEAE-cellulose ion-exchange chromatography. Scatchard analysis with corticosterone detected 30% fewer binding sites than a similar analysis with triamcinolone acetonide, probably because corticosterone binding was of lower affinity (Kd = 8.6 . 10(-9)M vs. 1.4 . 10(-9)M) and hence less stable. The relative glucocorticoid binding affinities of thirteen unlabeled steroids were obtained using either labeled steroid as ligand. Both ligands yielded similar results, suggesting that they both detected a similar binding site. The results suggest that AtT-20 cell cytosol contains a single class of binding site which detects both natural and synthetic glucocorticoids.


Melanoprotein: absence of a direct melanin-protein relationship in chick embryo melanocytes. Short-term synthesis of radioactivity labeled melanin (using DL-[2-14C]tyrosine or 2-[2-14C]thiouracil) by chick retinal pigment tissues in vitro was not influenced by inhibitors of protein synthesis, puromycin and cycloheximide. Co-ordinate synthesis of protein is, therefore, unnecessary for melanin synthesis, and melanoproteins must represent secondary interactions between melanin and protein. Melanin was isolated from chick embryo feather germs by extracting the proteins with hot dodecyl sulfate/mercaptoethanol. Melanin isolated from tissues incubated previously in L-[U-14C]valine medium had no associated radioactivity compared to the radioactivity of melanin prepared from tissues incubated in DL-[2-14C]tyrosine or 2-[2-14C]thiouracil. If melanoproteins exist at all, they are non-covalently bonded associations of melanin and melanosomal proteins.


Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors. Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were not dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.


2-Acetamidoglucal, a new metabolite isolated from the urine of a patient with sialuria. A new metabolite, namely 2-acetamidoglucal, has been found in the urine of a patient with sialuria in addition to the metabolites N-acetylneuraminic acid, N-acetylmannosamine, N-acetylglucosamine and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reported earlier. the structure has been identified by mass spectrometry and 360 MHz proton nuclear magnetic resonance spectroscopy and verified by synthesis. All accumulated compounds fit into the metabolic pathway for the biosynthesis of CMP-N-acetylneuraminic acid. Sialuria is discussed in terms of a failure of regulation of UDP-N-acetylglucosamine 2-epimerase.


Glucosyltransferase activity in kidney fractions of normal and streptozotocin-diabetic rats. Glucosyltransferase (UDPglucose: galactosylhydroxylysine-basement membrane glucosyltransferase), an enzyme specifically involved in collagen synthesis, was measured in various kidney fractions of normal, diabetic and underfed rats, using as basis the incorporation of radioactivity into protein during incubation with UDP[U-14C]glucose and alkali-soluble fetal calf-skin collagen. Three criteria of enzyme activity were compared: A, total radioactivity of the washed protein precipitate; B, this figure minus activity incorporated in the absence of the collagen acceptor; and C, radioactivity incorporated into the mixed amino acid fraction, collected by elution with dilute NH4OH from a Dowex 50 resin column after alkaline hydrolysis of the protein. Method A was found satisfactory using whole medulla or isolated glomeruli, since the average proportions of total protein radioactivity recovered in the NH3 fraction were 0.81 and 0.87, respectively, and the deviations were small. There was a larger and variable proportion of nonspecific incorporation using whole cortex. Incubation of a control set of sample without added collagen was found to be unnecessary (Method B). Per mg protein, medulla and glomeruli had more enzyme than did whole cortex. In diabetes, activity was enhanced in the 10,000 X g supernatant fraction of cortex, as previously reported. However, the increase associated with diabetes was even more consistent in the medulla, averaging 3-fold in the 10,000 X g pellet fraction. No increase was found in isolated glomeruli in diabetes. Also, no increase was seen in the kidneys of non-diabetic rats with body weight similar to that of the diabetics.


A comparison of the activities of aryl hydrocarbon monooxygenase in liver microsomes from mice of different strains during prolonged 3,4-benzo(a)pyrene administration. The content and activity of the components of liver microsomal aryl hydrocarbon monooxygenase system change biphasically during long-term 3,4-benzo(a)pyrene administration of C57BL/6  mice as well as to (C57BL/6 X DBA/2)F1 hybrids. The first activity peak (4--14 days) is associated with the induction of aryl hydrocarbon monooxygenase by 3,4-benzo(a)pyrene; the second peak (70--84 days) is related to noninductive mechanism. In DBA/2 mice, the second peak is absent while the slight increase in aryl hydrocarbon monooxygenase activity observed on days 14--28 indicates the aberrant inductive capacity of 3,4-benzo(a)pyrene under its prolonged administration. It is suggested that the weak sensitivity to the blastogenesis caused by 3,4-benzo(a)pyrene observed in C57BL/6 mice and in (C57BL/6 X DBA/2)F1 hybrids is due to the high level of liver aryl hydrocarbon monooxygenase activity at the time of tumor appearance.


Purification of leukocytosis-promoting substance from bovine parotid gland extract. A leukocytosis-promoting substance was purified from a crude bovine parotid gland extract. The purified substance was proved to be a single component by polyacrylamide gel disc electrophoresis. It stimulates an increase of peripheral leukocyte numbers in rabbits. The molecular weight of the physiologically active component was estimated to be 4.5 . 10(4), and the component was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be dissociated into two subcomponents.


Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein. Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.


A novel form of vitamin B-12 and its derivatives. A new isomeric form of cobalamins is reported. The conversion of cobalamin to cobalamin (the new form) is achieved by substituting the benzimidazole base by a less bulky group like H2O or CN- and modest thermal treatment. The back conversion of adenosylcobalamin to the corresponding regular form occurs in the "base-off" form at room temperature. It seems that the corrin ring becomes quite flexible in the "base-off" form and the freer axial movement of the cobalt atom flips the corrin ring into a different conformation. The change in conformation is borne out by subtle changes in the proton magnetic resonances on the corrin ring and the base, and very marked variation in the emission Mössbauer spectra. The latter is indicative of appreciable changes in the spatial conformation in the immediate vicinity of the central metal atom. The ultraviolet-visible and infrared spectra of cobalamin are indistinguishable from those of its corresponding regular form. The new conformational isomeric species is present as an impurity in all commercially available cobalamins (including pharmaceutical preparations). It raises the question whether the cobalamins' constitute the real biologically active anti-anemic factor in humans.


Light generation with Fenton's reagent. Its relationship to granulocyte chemiluminescence. A simple chemical system consisting of FeSO4 and H2O2 (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H2O2 was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH.) and singlet oxygen (1 deltagO2) quenchers suppressed chemiluminescence of the FeSO4 + tryptophan + H2O2 system; and, deuterium oxide (2H2O) enhanced chemiluminescence of both FeSO4 reactions. These observations suggest that a radical chain reaction involving both OH. and 1 deltag O2 is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H2O2. Since iron and H2O2 are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.


Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development. Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demonstrate the appearance of some factor(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase.  The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75 degrees C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.


Synergistic effects of thyroxine and glucocorticoid in induction of trypsin-like esteroprotease in mouse submandibular gland. The actions of thyroxine, 5 alpha-dihydrotestosterone and hydrocortisone singly or in combination in enzyme regulation in the submandibular gland were studied in intact and adrenalectomized female mice. 1. Adrenalectomy decreased the activity of trypsin-like esteroprotease (EC 3.4.4.-), and administration of hydrocortisone to adrenalectomized mice restored the activity to normal. 2. Hydrocortisone and thyroxine had synergistic effects in induction of esteroprotease in adrenalectomized mice, but 5 alpha-dihydrotestosterone and hydrocortisone did not have synergistic effects in either intact or adrenalectomized mice. 3. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was not influenced by change in the glucocorticoid level, but was increased by thyroxine and 5 alpha-dihydrotestosterone in both adrenalectomized mice and intact mice.4. Isoelectric focusing in polyacrylamide gel showed that there are three distinct activities of esteroprotease in this gland with isoelectric points of 5.6, 6.2 and 7.3. Both thyroxine and 5 alpha-dihydrotestosterone similarly induced these activities and glucocorticoids did not affected the isozyme patterns induced by the other two hormones.


Lysosomal enzyme secretion by macrophages during intracellular storage of particles. Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase beta-N-acetylglucosaminidase. Thus macrophage lysosomal enzyem secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both N-acetyl-beta-D-glucosaminidase and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.


The rate limiting step in the reticulocyte uptake of transferrin and transferrin iron. Effects of some incubation variables. Reticulocytes incubated in an isotonic NaCl saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticulocytes failed to remove completely the iron from the transferrin which they incorporated. It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unsepcific attachment of transferrin. At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.


Mutations altering the catalytic activity of a plant-type ribulose biphosphate carboxylase/oxygenase in Alcaligenes eutrophus. Use of a bacterial system has allowed the isolation of several mutationally altered species of ribulose-1,5-bisphosphate carboxylase/oxygenase having a wide range of catalytic activities. Biochemical and serological techniques were used to classify the mutant strains into three groups: (1) no detectable ribulose-1,5-bisphosphate carboxylase/oxygenase protein present (both subunits missing); (2) mutant strains having ribulose-1,5-bisphosphate carboxylase/oxygenases with lowered specific activities; and (3) mutant strains producing large quantities of catalytically inactive ribulose-1,5-bisphosphate carboxylase/oxygenase.


Insulin action in isolated fat cells. II. Effects of divalent cations on stimulation by insulin of protein synthesis, on inhibition of lipolysis by insulin, and on the binding of 125I-labelled insulin to isolated fat cells. The effects of ommission of Ca2+ and Mg2+ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca2+ + Mg2+)-free incubation medium incorporation of L-[14C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[14C]leucine incorporation only in the presence of added CaCl2 or MgCl2. Incubation of the cells in the (Ca2+ + Mg2+)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of 125I-labelled insulin to the fat cells was reduced in the absence of Ca2+ and Mg2+ but was not abolished, even in the presence of EDTA. Ca2+ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg2+, Sr2+ and Ba2+. Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.


Folates of rat tissue. Bioassay of tissue folylpolyglutamates and a relationship of liver folypolyglutamates to nutritional folate sufficiency. The folate content of young rat tissues extracted into boiling ascorbate was assayed by Lactobactillus casei both without and after treatment by a folate-free preparation of conjugase. The total folate content of various tissues was: liver, 8.9 microgram/g; kidney, 2.6; adrenal, 2.6; bone marrow, 2.4; spleen, 0.9; erythrocytes, 0.8; small intestinal mucosa, 0.7; small intestinal smooth muscle, 0.8; heart, 0.6; brain, 0.4, and skeletal muscle, 0.1 microgram/g tissue. For most tissues, with the exception of muscle and kidney, approximately 80% of the total folates assayed as longer chain length folylpolyglutamates. When liver folates were analyzed from rats fed folate-supplemented, control and folate-deficient diets, a relationship was found between folate nutrition and distribution of folylpolyglutamates. The proportion of total folates in the form of longer chain length folylpolyglutamates was greatest in the livers of folate-deficient rats and least in the livers of folate-supplemented rats.


The dimerization of ferrihaems. IV. Studies on haematoferrihaem and a general appraisal of the nature and implications of dimerization. The dimerization of haematoferrihaem was studied in phosphate buffer in the pH range 7.02--8.14. The absorbance of dilute solutions decreased over a period of several hours due to adsorption of haematoferrihaem to glass vessels. This problem was overcome by using dilute solutions within 10 min of preparation. Spectrophotometric data were consistent with a dimerization process according to the equation 2 monomer in equilibrium dimer + H+ as found earlier for other ferrihaems studied. The value of K, defined as K = [dimer] [H+]/[monomer]2, was found to be 1.00 . 10(-2). Rate constants for the forward and reverse steps in dimerization were determined at pH values of 6.63, 7.01 and 7.44, using the temperature-jump technique. The reaction pathway for dimerization was found to be similar to those of deuteroferrihaem and mesoferrihaem, but different from that of coproferrihaem. A general appraisal of the factors influencing dimerization is attempted in the light of accumulated data on various ferrihaems. With the exception of protoferrihaem, it is suggested that dimerzation increases with the hydrophobicity of the 2,4 substituents. The additional stability of the protoferrihaem dimer is explained in terms of increased interaction due to conjugation of the vinyl groups with the porphyrin macrocycle.


Hydrolysis of uridine diphosphate N-acetyl-D-glucosamine by embryonic cells of the hamster and rat. Determination by high-pressure liquid chromatography. The hydrolysis of UDP-N-acetylglucosamine by extracts of rat embryo cells has been compared to the hydrolysis of this sugar nucleotide by extracts of hamster embryo cells. A method utilizing high-pressure liquid chromatography was developed to separate and quantify the products of the hydrolysis. Rat embryo cells as well as hamster embryo cells hydrolyzed UDP-N-acetylglucosamine virtually completely under the experimental conditions. The principal product of the hydrolysis of UDP-N-acetylglucosamine by hamster embryo cells was N-acetylglucosamine. The sugar moiety of N-acetyglucosamine, produced by hamster embryo cells, was identified by thin-layer chromatography to be glucose. By contrast, the major metabolite (60--70%) of the hydrolysis of UDP-N-acetylglucosamine by rat embryo cells was identified as a phosphorylated acetylamino sugar. Only about 30--40% of the substrate was degraded to the free acetylamino sugar. Examination of the sugar moiety of the phosphorylated and of the free acetylamino sugar by thin-layer chromatography indicated that it was a mixture of glucose and of the epimers, mannose or galactose. The identity of the epimers has as yet not been established. Thus, unlike hamster embryo cells, rat embryo cells contain an active epimerase. However, the phosphohydrolase of the rat embryo cell which degrades the phosphorylated acetylamino sugars to the free acetylamino sugars seems to be less active than the enzyme in the hamster embryo cell. On the basis of the data of this study a comparison of the pattern of hydrolysis of UDP-N-acetylglucosamine by normal and transformed embryonic cells of the rat and of the hamster appears feasible.


Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen, antiestrogen and progesterone administration. A synthetic progestin, 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione (ORG 2058), was utilized to measure progesterone receptors from the rabbit uterus. This steroid has a high affinity for both cytosol and nuclear receptors, with KD values of 1.2 nM (at 0--4 degrees C) and 2.3 nM (at 15 degrees C), respectively. Administration of estradiol-17 beta or a non-steroidal antiestrogen, tamoxifen, for 5 days to estrous rabbits led to a progressive rise in the cytosol receptor levels: from 34,000 to 120,000 (estradiol-17 beta) and 80,000 (tamoxifen) receptors/cell, without any major influence on the nuclear receptor content. A single intravenous injection of progesterone (5 mg/kg) elicited a 3-fold increase in the mean nuclear receptor content at 30 min after injection (from 18,000 to 48,000 receptors/nucleus). Nuclear receptor accumulation was short-lived and returned to control levels within 4 h after treatment. A second dose of progesterone given 24 h later doubled the nuclear receptor level (from 18,000 to 35,000 receptors/nucleus). The concomitant decline in the cytosol receptor content was twice that accounted for by the nuclear receptor accumulation (70,000 vs. 30,000, and 40,000 vs. 17,000 receptors/cell, after the first and second progesterone injection, respectively). Following progesterone administration, the cytosol receptor level reached a nadir by 30 min, exhibited minimal replenishment within the ensuing 24 h, and remained at approx. 50% of the pretreatment values. After a single dose or two consecutive doses of progesterone, total uterine progesterone receptor content declined to about 60% of the level prior to each dose, a nadir being reached at 2 h after treatment.


Alanine metabolism in skeletal muscle in tissue culture. Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L6) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P less than 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P less than 0.0005) as well as medium (r = 0.50, P less than 0.025) and intracellular (r = 0.85, P less than 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P less than 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rater to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.


Inhibition of phorbol ester-accelerated amino acid transport in bovine lymphocytes. 12-O-Tetradecanoylphorbol-13-acetate, a highly active tumor-promoting agent and lymphocyte comitogen, rapidly accelerates the transport of alpha-aminoisobutyric acid in cultured bovine lymphocytes. Structure-activity studies show that the ability of phorbol diesters to accelerate alpha-aminoisobutyric acid uptake runs parallel to their potency as lymphocyte comitogens and as tumor promoters in mouse skin. This phorbol ester-accelerated, amino acid transport is largely insensitive to the inhibition of RNA and protein synthesis by actinomycin D and cycloheximide, respectively, and is insensitive to the inhibition of membrane movement by cytochalasin B and colchicine. Retinoic acid, an antagonist of the tumor-promoting and comitogenic actions of phorbol esters also inhibits the acceleration of amino acid uptake by 12-O-tetradecanoylphorbol-13-acetate; however, the epoxy derivatives of retinoic acid and structurally related analogs, which are potent antagonists of the other aspects of phorbol ester activation of lymphocytes, are inactive in blocking amino acid uptake. Comparative studies in lymphocytes show that this phorbol ester elicits a number of metabolic responses which appear to originate at the cell membrane and that these are differentially antagonized by retinoic acid, the 5,6-epoxide of retinoic acid, and related retinoid analogs.


Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine. It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH+4 and/or H+ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH4Cl-induced acidosis were prepared. These extracts were tested for their effect on NH+4 excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH+4 excretion in the toad urinary bladder. We found no effect on H+ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH+4. The factor could act as a permease in the bladder cell or as a stimulator of an NH+4 transport system.


Transport of L-carnitine induced by prednisolone in an established cell line (CCL 27). A possible explanation of the therapeutic effect of glucocorticoids in muscular carnitine deficiency syndrome. Prednisolone (10(-8)--10(-5) mol/l) in the growth medium for 24 h increased the rate of uptake of L-[3H]carnitine in an established cell line (CCL 27) to 164 +/- 6% (mean +/-S.E.) of the rate observed in untreated cells. At the same time the intracellular content of free L-carnitine increased about 20%. The simultaneous addition of prednisolone (10(-6) mol/l for 24 h) and L-carnitine (10(-4) mol/l for 96 h) to the growth medium increased the rate of uptake to 225 +/- 8% (mean +/-S.E.) of that in untreated cells. The increase seemed to be mediated through an increase in number of carriers, as judged by the increase in V of the transport process with unchanged Km. Phosphodiesterase I, an enzyme mainly localized in the plasma membrane, increased its activity about 3.5 times when cells were stimulated with prednisolone. Thus, it seems that the increase in the rate of uptake of L-carnitine mediated by glucocorticoids, is part of a more general effect on the plasma membrane. The observations offer an explanation to the observed clinical improvement in patients with muscular carnitine deficiency treated with glucocorticoids and/or L-carnitine.


[IR-spectroscopic study of the temperature anomaly of water sorbed onto DNA]. IR-spectra of liquid water and that sorbed on native and denaturated DNA at different relative humidity (RH) are obtained in the region of valent OH-(OD)-oscillations within the temperature range 0--100 degrees C. An S-shape decrease of absorption intensity was observed for water sorbed on DNA at 94% RH in two temperature regions (25--45 and 75--95 degrees C). Proceeding from the assumption that individual absorption bands are described by equal symmetrical functions, development of complex absorption contour of D2O in the region 3000--2000 cm-1 into constituents was performed. It enabled to determine band parameters of underphase vuf and synphase vsf valent and overtone of deformation oscillations of OD-groups in liquid and sorbed water and to reveal at higher temperatures the bands of "free" OD-groups (v = 2668 cm-1). Relative contribution of integral intensity of the latter at 96 degrees C in sorbed water is about 4 times lower than in the liquid one.


[Role of singlet-excited and triplet states in chlorophyll photooxidation]. Effect of p-benzoquinone concentration [Q] on the yield of ion-radicals during photooxidation of chlorophyll a with p-benzoquinone was studied by the method of impulse photoconductivity. It has been shown that in the studied range [Q] = 10(-6)--10(-1) M the relationship delta n ([Q]) has a bell-like shape with the maximum at [Q] approximately 10(-3) M. From the relationship obtained the ratio beta = delta ns/delta nt of the contributions of singlet--excited (Chl) and triplet (Chl) states of the pigment into the total yield of ion-radicals is evaluated: for [Q] less than or equal to 10(-1) M beta less than or approximately 10(-3). Primary contribution of the triplet state to the formation of ion-radical is explained, first, by a larger, as compared to the singlet-excited state, life time of the state Chl in solution and second, by the fact that the ion-radical pair 3(Chl+Q-) in the triplet state regenerates less effectively to Chl+Q, than the pair (Chl+Q-).


[Effect of intramolecular and intermolecular reactions of the intensity of histone H2A and H4 fluorescence]. Fluorescence of histones H2A and H4 of homo- and hetero-complexes has been studied. It has been found that in the composition of small aggregates consisting of several molecules, the change of histone fluorescence intensity is caused by the change of their tertiary structure. In the composition of large aggregates, including several dozens of molecules, the intramolecular interactions contribute to the change of histone fluorescence. The intermolecular histone interactions are specific and have a cooperative character.


[Determination of the kinetic association and dissociation constants of proteins]. On the basis of protein solution characterising by a reversible association--dissociation monomer--n-mer without intermediate products the statistic moments have been calculated. On the basis of these expressions and numerical experimental values one can compute the values of kinetic constants of the association--dissociation reactions.


[Effect of Ca2+ on glucose penetration through the pink ghosts of human erythrocytes]. It has been found that in the presence of millimolar concentrations of calcium added into pink shadows of erythrocytes (C1/2 = 4 mM) the velocity of glucose transport increases three-fold as compared to control shadows. Calcium added from the outside does not affect the glucose transport. Intracellular calcium induces a structural transition in the erythrocyte membrane, which is revealed by spectrin aggregation (electrophoretic data). Study of temperature relationships of glucose transport velocity in the presence of different concentrations of intracellular calcium as well as of the effect of inhibition of transport with phloricin under this condition allows a conclusion that due to Ca-induced transition in the membrane new specialized carriers get involved into glucose transport.


[Effect of toxins from the scorpion Buthus eupeus on the sodium channels of the membranes of nodes of Ranvier]. Effect of three toxins M3, M7 and M8 from Buthus eupeus poison on sodium channels of Ranvier node membrane of the frog was studied under potential fixation. Toxin M3 was found to produce no effect on sodium currents of Ranvier node membrane of the frog. Inactivation kinetics difference between normal and modified sodium channels was used in estimating a fraction of toxin-modified channels. The toxins were found to interact with the channels in the ratio 1 : 1, the dissociation constants for M7 and M8 equalling 1.7 . 10(-7) M and 0.9 . 10(-9) M respectively. Dependence of the effect of scorpion toxins on the membrane potential is stated.


[Magnetic relaxation of water protons and the state of the water photodissociation system in chloroplasts]. The spin-spin relaxation time T2 of water protons is measured in chloroplast suspensions. It is shown that chloroplast lamellae efficiently increase the rate of proton magnetic relaxation, this effect being a linear function of chloroplast concentration. There is a fast exchange between the water molecules in different environments, thus the measured relaxation rate is a weight average of all water molecules. The rate of proton relaxation is decreased when membrane bound manganese is removed from chloroplasts by tris-washing procedure. The relaxation effect is slightly reduced in the presence of Photosystem II electron acceptors.


[Effect of paramagnetic additives on the spin lattice relaxation of the protons of intracellular water]. Studies of the effects of Mn2+ and Fe3+ paramagnetic ions on spin-lattice relaxation of water protons in plant cells have shown that paramagnetic admixtures which are in the cells under normal conditions (in natural quantities) produce no significant effect on the rate of spin-lattice relation of water protons. On the basis of the investigations of proton, spin-lattice relaxation at increased content of Mn2+ paramagnetic ions in plant cells possible separation of relaxation times T1l and T1s which are corresponded by different water fractions in the cells is shown as well as possible stimation of water quantity in the fractions.


[Electrical interaction between muscle fibers]. When one zone of motor nerve endings is "switched off" by injecting d-tubocurarine solution into it the propagation velocity of action potentials in different regions of biarticular muscles is changed. In the middle regions of the muscle where the interference of two opposite waves of electric activity in most clearly pronounced, the "switching off" of the proximal zone of motor endings decreases the AP propagation velocity in the muscle fibres of the distal zone. When the distal zone of motor endings is "switched off" the AP propagation velocity increases. The data obtained point to the effect of electric processes in some muscle fibres on those in other muscle fibres. The degree of this effect is determined by the muscle architectonics and topography of motor nerve endings.


[Spectral forms of the chlorophyll of mutants of Chlamydomonas with inactive photosystems]. Investigations of non-photosinthesizing mutants of Chlamydomonas reinhardii with damaged activity of both or one of the photosystems have shown that the chlorophyll a form with the absorption maximum at 685 nm and low temperature fluorescence band at 696 nm is a part of the pigment-protein complex of PS-2. Chlorophyll a forms with absorption maxima at 689, 698 and 703 nm belong to the pigment-protein complex of PS-1. They seem to be responsible for the long-wave band of chlorophyll fluorescence with the maximum at 707--718 nm. Chlorophyll a forms with absorption at 661, 667 and 678 nm and a short-wave fluorescence band at 685--690 nm rank among the pigments of the light-converging complex, as well as chlorophyll b with the absorption maximum at 644 and 649 nm.


[Mechanical deformations of bioconstructions during different stages of morphogenesis]. The stress-deformation regularities of the body behaviour are taken as principal pattern formation factors in bioconstructions: mutually compressed cap rays of Acetabularia and contiguous tissue layers (the model). With the growth of rays or layers mechanical tensions which are initiated in them increase. If no morphogenetic loss of shape stability is resulted, an alternative algorithm of natural complication of their initial pattern is finally realized and ruptures, shifts and other deformations are formed in some weakened elements of the constructions. Among rigid stable parameters of biomaterials and geometric parameters of bioconstructions only the latter prove to be the determining ones in feasibility criterion of this or that extreme case of pattern formation (with the loss of stability or fractures). Restrictions imposed to the values of possible deformations are conditioned in case of the loss of pattern stability by geometric parameter, in case of fractures--by the strength parameters.


[Effect of specialized contacts on fiber interaction during the spread of excitation in smooth muscle and myocardial tissues]. A mathematical model of closely packed parallel muscle fibres with side by side junctions was studied. High coupling coefficient for both heart and smooth muscle fibres was shown to be available at rather low junction permeability. The coupling coefficient for heart fibers is equal to 0.5 at specific junction membrane resistance as high as 100 omega cm2. For the same coupling ratio in smooth muscle the specific junction membrane resistance should be even greater (1000 omega cm2). The role of nexuses and tight junctions in conduction of excitation is discussed.


[Theory of EEG potential in a model of the thin membranes of the brain]. The methods of estimating a momentary value of the epidural potential using several values of EEG, sampled at the same instant, are explored theoretically. The weights of the summation of these EEG-potentials for the case of simple disposition of electrodes, are single-valued by means of elementary consideration of the potential theory and symmetry. The space filters of EEG obtained in such a way, were analysed on the basis of the model of thin integuments of the brains. The systems of 4--8 electrodes on the scalp are proposed for the estimation of mono- and bipolar ECoG's, which are less sensitive to ECoG of the remote cortex than ordinary derivations of EEG.


[Mechanism of the effect of neuronal response suppression in the auditory nerve during exposure to bitonal stimuli]. It is supposed that interreceptor currents existing due to microphonic and summating potentials are the exiting factors for the neuron endings of the cochlear nerve. A model is proposed of the formation of the cochlear neuron responses under the stimulation of the hearing system by a tone signal with a changing frequency and by a signal consisting of two tones. It is shown that the proposed model explains the effect of inhibition of the neuron response to a given tone signal when another tone signal of proper intensity arises in a definite frequency region.


[Biosublicons].

[Biosublicons]. Biosublicon is an ice cylindrical thread on the surface of which a continuous helix of chain biopolymers is packed. Such structures are spontaneously formed during sublimation of frozen aqueous solutions of biopolymers with simultaneous illumination (lambda greater than 220 nm). Biosublicons are produced from diluted solutions containing a racemate mixture of amino acids and nucleotides. Their peculiar formation is discussed.


[Computer study of the dynamics of human movement]. Dynamics of control and construction of man's movement was modeled on a computer. Measuring--computing complex was elaborated which involved means for movement recording, mathematical model and program provision for computing direct and inverse problems of dynamics. Equations of movement and subsequent operations were carried out directly on the computer. Cubic smoothing splines were used during approximation and differentiation of the experimental data. Two human concrete movements were studied. It was found that control over man's elementary movements was realized according to TAU laws in complex movements controling and stabilizing functions of the movements can be distinguished.


[Calorimetric study of heat denaturation of beta-trypsin]. Thermal denaturation of beta-trypsin was studied by scanning microcalorimetry. It is shown that the denaturational transition is well described by the one-step model of transition between two macroscopic states. The specific enthalpy of denaturation is a linear function of temperature, the extrapolation to 110 degrees leading to a value of 54.2 J/g, i. e. to the value being characteristic for small compact globular proteins.


[Internal movement within globular proteins in accordance with the relationship between temperature and spin-lattice relaxation time]. The minima on the temperature dependence of T1 for protein protons were observed in dried SA at temperature about -100 degrees C and also about 0 degree C in SA and RNAase solutions in D2O. The minima of the temperature dependence were ascribed to motions of methyl groups of proteins, in the first case, and to oscillation of the polypeptide chain in the second case, both at characteristic frequencies of 10(8) s-1 in the minimum points.


[Effect of creatine phosphate synthesis on the membrane potential of mitochondria]. The method of the penetrating ions allows to observe the creatine phosphokinase function of heart mitochondria in steady state conditions. The addition of ADP to respiring mitochondria decreases the membrane potential in the course of the ATP synthesis. The subsequent addition of creatine decreases the membrane potential for a long period of time because of ADP formation in creatine phosphokinase reaction. It was shown in the experiments with maintainance of the constant concentration of ADP or ATP in the medium that the ADP concentration in the medium acts mainly on the membrane potential during the creatine phosphate synthesis. There is no effect of creatine on the membrane potential of liver mitochondria which have no creatine phosphokinase.


Chromosome survey of newborn infants in Tokyo: follow-up study for XYY. In order to ascertain the frequency of chromosome aberrations among newborn infants in Japan, a chromosome survey of a large number of newborn infants is in progress. A series of 10,270 consecutive newborn babies, 5,341 male and 4,929 female, have been screened for clinical manifestations of autosomal aberrations and for sex-chromatin and sex-chromosome aberrations. Chromosome studies were carried out on 185 infants with suspected chromosome aberrations. Of these, 23 had abnormal karyotypes, including 2 males with a 47,XXY complement, 1 female with 45,X complement, 3 males with a 47,XYY complement, 2 with trisomy 13 syndrome, 3 with trisomy 18 (including one mosaicism), 10 with Down syndrome (including 1 mosaicism), 1 with B5p partial trisomy, and 1 with Y-D translocation. Developmental studies of four XYY children and one 45,X girl are in progress.


Platelet alpha granules contain a growth factor for fibroblasts. Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha-granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta-thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta-thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.


Distribution of iron between the binding sites of transferrin in serum: methods and results in normal human subjects. When it is incompletely saturated with iron, transferrin may exist in four molecular forms: apotransferrin, monoferric (A) transferrin (with iron occupying only the A site of the protein), monoferric (B) transferrin, and diferric transferrin. By combining electrophoresis in urea-polyacrylamide gels with crossed immunoelectrophoresis using specific antihuman transferin antiserum, it is possible to display and estimate the concentration of each of these four forms in normal human serum. The distribution of iron between the binding sites of transferrin is neither random nor determined by the relative binding strengths of transferrin's two sites. Rather, the more weakly binding and acid-labile B site of the protein is predominantly occupied.


Presence of mast cell precursors in peripheral blood of mice demonstrated by parabiosis. The presence of mast cell precursors in peripheral blood was examined. The beige C57BL/6 (bgj/bgj, Chediak-Higashi syndrome) mouse was parabiosed with the normal C57BL/6 mouse. Mast cells containing giant granules and originating in the beige partner appeared in the normal parabiont. A comparable proportion of normal-mouse-type mast cells developed in the beige parabiont as well. In spite of the low radiosensitivity of mature mast cells, irradiation of the normal parabiont reduced the proportion of normal-mouse-type mast cells appearing in the beige partner. It was concluded, therefore, that the precursors of mast cells can migrate through the bloodstream.


Preferential inhibition of murine macrophage colony formation by prostaglandin E. Murine bone marrow progenitor cells that gave rise to macrophage colonies in semisolid agar were found to be more sensitive to prostaglandins of the E series (PGE) than were precursor cells of granulocytes and megakaryocytes. Macrophage colonies themselves had different sensitivities to the molecule. Precursor cells of macrophages that formed colonies in the presence of a stimulating activity from L cells (L-cell CSA) were inhibited to 50% levels by 3 x 10(-9)-M PGE. Macrophage progenitor cells, which require both L-cell CSA and rat hemolysate for colony growth, were inhibited to the same level by 3 x 10(-7)-M PGE. Other colony types (granulocytes and megakaryocytes) were sensitive to PGE only at concentrations greater than 10(-6) M. Accordingly, addition of different PGE concentrations to the culture assay should allow easy detection of precursor cells with morphologically distinct end cells. The different sensitivities to PGE of two macrophage colony types of different maturation stages indicate that PGE may provide feedback to control macrophage formation by inhibiting proliferation and differentiation of immature monocytoid cells.


Increased circulating neutrophils with surface receptor activity for immunoglobulin G in polycythemia vera and myeloid metaplasia. Reports of heterogeneity of IgG receptor activity of normal circulating neutrophils prompted measurements in myeloproliferative disease to determine if dysplasia of the hematic stem cell resulted in an abnormality of this membrane property. IgG receptors were assayed by rosette formation in suspension with human Rh-positive erythrocytes sensitized with high-titer Rh antiserum. IgG receptors were detected on 19 +/- 1.6% (mean +/- SEM) of neutrophils from 45 normal subjects. A significant increase in IgG-receptor-bearing neutrophils was found in polycythemia vera (PV) and myeloid metaplasia (MyM), with values of 70 +/- 3.6% and 69.7 +/- 4.3%, respectively. Normal values were observed in polycythemic states not due to myeloproliferative disease and in chronic myelocytic leukemia. Rosette-forming neutrophils were increased to 52.3 +/- 3.7% in infection and inflammatory disease, but this value was significantly lower than those in PV and MyM. Increased IgG receptors in PV and MyM may be related to the activated state of the neutrophil and may result from an intrinsic cellular abnormality of the proliferating clone or from altered bone marrow release. Quantitation of neutrophil IgG receptors may be of value in the differential diagnosis of PV and MyM and may offer insights into the derangement of hematopoiesis that underlies these myeloproliferative disorders.


Fetal hemoglobin in polycythemia vera: cellular distribution in 50 unselected patients. Fetal hemoglobin was studied in 50 unselected patients with polycythemia vera with chemical determinations of Hb F and measurements of F-cell levels using fluorescent anti-Hb F antibodies. Although in the majority of the patients Hb F production did not differ from that in normal controls, in 20% of the patients F-cell values were above the normal range. There was no correlation between F-cell values and duration of disease, treatment modality, presence of myelofibrosis, or hematologic parameters at the time of study. In 5 of 50 patients 30%--45% of the erythrocytes contained Hb F; such striking elevations of F cells may reflect a distorted differentiation of erythroid precursor cells in certain patients with this disorder.


In vivo erythropoietin requirements of regenerating erythroid progenitors (BFU-e, CFU-e) in bone marrow of mice. Erythroid progenitors (B-8, B-4, CFU-e) in the femoral marrow of polycythemic mice were measured by in vitro culture assays after a single administration of BCNU or Myleran. BCNU reduced pluripotent stem cells to 40% and erythroid progenitors to less than 5% of normal. B-8, the earliest erythroid progenitors, regenerated without erythropoietin (Epo) completely within 5 days. At 14 days after BCNU, intermediate progenitors (B-4) attained 60% of their normal numbers and CFU-e attained approximately 30%. Daily injections of Epo promptly restored normal B-4 numbers and near-normal CFU-e numbers in BCNU-treated mice. After Myleran, CFU-s remained below 2% of normal for 14 days, and no regeneration of the B-8 occurred with or without daily Epo injections. The findings suggest that regneration of B-8 was dependent on cell inflow from the pluripotent stem cell compartment but was independent of the presence of Epo. Intermediate progenitors (B-4) required Epo and the presence of B-8 for complete and permanent regeneration. CFU-e were the most Epo-dependent of the three progenitors. B-4, recruited by Epo, required after their formation a second exposure to the hormone in order to progress into the CFU-e stage.


In vitro assay for erythropoietin: erythroid colony formation in methyl cellulose used for the measurement of erythropoietin in plasma. Erythroid colony formation in methyl cellulose has been used for the measurement of erythropoietin in plasma. Livers from newborn mice less than 24 hr old were found to provide convenient target cells. Newborn mouse liver contains a substantial number of erythroid colony-forming cells (CFU-e) that have a high sensitivity to erythropoietin, the dose--response curve for erythropoietin reaching a plateau at 50 mU/ml. As little as 0.5 m/ml of the hormone is detectable. Removal of cells that adhered to glass prior to culturing doubled the number of colonies formed in the presence of erythropoietin. Addition of untreated plasmas that showed high erythropoietin titers in the exhypoxic polycythemic mice assay gave variable results. Some of the plasmas stimulated colony formation actively and in a linear fashion. However, the majority of the plasmas were toxic to the cultures. Dialyzing the plasmas for 3 days against distilled water effectively removed the toxicity. Results obtained with the method are in good agreement with the values found using the exhypoxic polycythemic mice assay.


Lymphocyte-mediated lysis of antibody coated human red cells in the presence of human serum. When peripheral blood lymphocytes and human red cells coated with IgG were incubated in vitro in culture medium, antibody-dependent lymphocyte-mediated lysis was observed. This lysis was markedly inhibited by the addition of purified monoclonal IgG1 (1000 microgram/ml) to the culture medium. In contrast, lysis by lymphocytes of sensitized red cells in the presence of undiluted human serum was equal to or greater than lysis in medium alone, even in the presence of IgG1 at 1000 microgram/ml, despite the high concentration of IgG in human serum (6000--19,000 microgram/ml). Serum heated to 56 degrees C for 30 min also restored lysis in the presence of IgG1. When serum was separated into three fractions by passage through a Sephadex G-200 column, the third fraction, which contained proteins with a molecular weight of less than 100,000 d (but neither of the other two fractions nor purified human albumin), restored lymphocyte-mediated lysis in the presence of IgG1.


Erythropoietin-dependent primary pure erythrocytosis. We investigated the pathogenesis of isolated erythrocytosis of 14 yr duration in a 28-yr-old man. The increase in red cell mass was attributed to increased erythropoietin production. An extensive search for recognized causes of secondary erythrocytosis was unrevealing. Family members were found to be hematologically normal. After reduction of the circulating red cell mass by 20%, erythropoietin activity nearly quadrupled, thus suggesting a normal erythropoietin response to phlebotomy. When bone marrow cells of the patient were cultured in plasma clots in the absence of added erythropoietin, endogenous erythroid colony formation was observed, a pattern previously believed to be specific for polycythemia vera bone marrow cells. Our observations suggest that the erythrocytosis in this individual is best explained by an abnormal "servoregulatory" mechanism of erythropoietin production. In addition, this is the first instance in which the rule that endogenous erythroid colony formation is correlated with the diagnosis of polycythemia vera has not held.


Separation of erythroid progenitor cells in mouse bone marrow by isokinetic-gradient sedimentation. Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone-dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin-dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.


Improved survival of increased-risk myeloma patients on combined triple-alkylating-agent therapy: a study of the CALGB. Two hundred fifty-two previously untreated evaluable patients with multiple myeloma were entered into a study testing a regimen of three intravenous alkylating agents, melphalan, cyclophosphamide, and carmustine (BCNU), given in combination (BCMP) against a regimen employing oral melphalan (MP). Both regimens included a tapering course of prednisone. Objective responses based on the Myeloma Task Force criteria were significantly more frequent in the group receiving BCMP. Survival for the entire group of BCMP-treated patients was not significantly better than that for MP-treated patients (p = 0.62). However, when the survival of the poor-risk (high tumor cell load) group of patients treated with BCMP was compared with the survival of the poor-risk (high tumor cell load) group of patients treated with MP, an improvement in survival attributable to BCMP therapy was seen (p = 0.049 and 0.02, respectively). In the good-risk (low and intermediate tumor cell load) group, BCMP treatment resulted in a trend toward poorer survival, but this did not achieve statistical significance (p = 0.080 and 0.23, respectively). These results indicate that optimal therapy in myeloma may be dependent on the extent of disease at the time of first treatment. Additional studies to explore the effects of treatment intensity and duration are needed in order to design improved myeloma treatment based on the patient's extent of disease.


Contamination of erythropoietin by endotoxin: in vivo and in vitro effects on murine erythropoiesis. Endotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU-e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10(-5)-10(-3) mug of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.


Fetal characteristics of erythrocytes in sickle cell anemia: an immunofluorescence study of individual cells. In a group of disease states that includes sickle cell anemia (SS disease), two fetal erythrocyte markers, Hb F and i antigen, persist into adulthood. Using the technique of single-cell immunofluorescence, we determined the expression of l-i antigens and the presence of Hb F within populations of erythrocytes. Subjects tested included normal adults, normal newborns, patients with SS disease, and individuals with sickle cell trait. We classified erythrocytes reacting to anti-i as i cells and those reacting to anti-l as l cells, a terminology analogous to that used to identify erythrocytes containing increased amounts of Hb F as F cells. The expression of l and i antigens within populations of both normal and SS erythrocytes was found to be heterogeneous. The proporations of both i cells and l cells in all SS patients studied exceeded those found in normal adults, and an overall stronger-than-normal reactivity of individual SS cells to the two antibodies was observed. Proportions of F cells showed no correlation with proportions of i cells; and with double fluroescence staining for both Hb F and i, a significant proportion of each total SS red cell population was found to carry only one or the other marker. These findings confirm and clarify on a cellular level our previous demonstration, by means of quantitative hemagglutination, that there is increased expression of both l and i by whole populations of SS erythrocytes. In addition, we provide here new information on the expression of l and i within populations of normal human erythrocytes.


Characteristics of murine megakaryocytic colonies in vitro. Characteristics of murine megakaryocytic colonies and their progenitor cells (CFU-m) were studied in vitro in agar gel. Colony growth required the presence of poke-weed-mitogen-stimulated spleen-conditioned medium. The number of colonies formed was linearly related to both the number of marrow cells plated and the amount of conditioned medium added. In addition, CFU-m were found in both the spleen and peripheral blood. Conditioned medium was also made without plasma, and this resulted in a cloning efficiency greater than that of conditioned medium prepared with plasma. The percentage of CFU-m in DNA synthesis was low (10%), as determined both in vivo and in vitro. Velocity sedimentation revealed that the majority of CFU-m sedimented at 4.3 mm/hr and had a tritiated thymidine (3H-TdR) suicide rate of 1.5 +/- 1.5%. A shoulder on the profile of CFU-m sedimented at approximately 6 mm/hr, with a suicide rate of 79 +/- 2%. Analysis of these data indicated that the majority of CFU-m were not in cycle or were in a long G1 period. The results suggest that CFU-m is a primitive progenitor, possibly closely related to murine splenic colony-forming units (CFU-s), analogous to erythroid bursts and granulocytic colony-forming units.


Coculture studies of 16 untransfused patients with aplastic anemia. We studied the effects of peripheral blood lymphocytes from 16 untransfused patients with severe aplastic anemia (AA) of diverse etiologies on the growth of granulocyte-macrophage colonies from normal marrows. Normal lymphocytes in our system increased the number of granulocytic colonies by 31 +/- 6% (mean +/- SEM). Lymphocytes from 3 of 16 untransfused AA patients significantly inhibited growth in HLA-matched sibling marrows (-30%, -40%, and -37%; p less than 0.01). Although these results suggest that the majority of cases of AA are not mediated by a coculture-detectable immunologic mechanism, studies using lymphocytes obtained from AA patients before transfusions may detect the subpopulation whose disease is immune-mediated and who may therefore respond to immunosuppressive therapy.


Granulocyte concentrate function during preservation: effect of temperature. Granulocyte concentrates collected from normal donors are necessarily stored for varying intervals up to the time of transfusion. However, information regarding the fate of collected cells and the optimal mode of storage in vitro in the interval between collection and transfusion is far from complete. We studied granulocyte function during preservation of granulocyte concentrates for up to 72 hr. The initial and most consistent alteration in granulocyte function during storage was failure of random migration and chemotaxis after 24 hr of storage (50% and 61% of normal, respectively). By 48 hr the respiratory burst was decreased by 42%, whereas at 48 hr phagocytic and bactericidal activities were nearly normal. Defects in migration and respiratory burst are not due to delayed activation of these functions but to absolute decreases in maximum rates of migration and oxygen consumption. Comparison of granulocyte concentrate storage at 6 degrees C versus room temperature indicated at 24 hr an improved (p greater than 0.02) but still abnormal (p greater than 0.02) chemotactic response with 24 degrees C storage and at 48 hr no difference in migration but a slight advantage in bacterial killing at 6 degrees C storage. These studies show that severe impairment of granulocyte function occurs within 24 hr of collection by centrifugal means; consequently, granulocyte concentrates should be transfused as soon as possible after collection.


In vitro cytotoxic response to human myeloma plasma cells by peripheral blood leukocytes from patients with multiple myeloma and benign monoclonal gammopathy. Peripheral blood leukocytes (PBL) from myeloma patients were studied for their capacity to lyse plasma cells from myeloma patients, benign monoclonal gammopathy (BMG) patients, and nonneoplastic disease patients. Plasma cells were isolated from bone marrow, labeled with 51Cr, and cultured with PBL isolated from patients with myeloma, BMG, or nonneoplastic disease, as well as normal individuals. PBL from patients with multiple myeloma demonstrated responses to autologous or allogeneic myeloma plasma cells. Optimum conditions for cytotoxic response included a responder-to-stimulator ratio of 1:1 and an effector-to-target ratio of 20:1. PBL from normal individuals or patients with BMG failed to demonstrate this response. However, PBL from BMG patients, but not normal individuals, could be induced to kill myeloma plasma cells (but not nonmyeloma plasma cells) by simultaneous stimulation with allogeneic lymphocytes and myeloma plasma cells.


Transport accounts for glutathione turnover in human erythrocytes. Human erythrocytes were incubated with 3H-glycine to label the glutathione pool. These cells were then used to determine the rate of oxidized glutathione (GSSG) transport out of erythrocytes. For 6 normal individuals, the mean transport rate was 6.7 nmole GSSG/hr/ml red cells. This transport rate would suggest a half-life of 4.7 days for the erythrocytic glutathione, which is in close agreemwnt with the observed in vivo half-life of 4 days. These data demonstrate that GSSG transport can account for the observed turnover of erythrocytic glutathione.


Erythropoietin levels in the course of a patient with erythropoietin-producing renal cell carcinoma and transplantation of this tumor in nude mice. Erythropoietin was measured by exhypoxic polycythemic mouse method in the course of a 64-yr-old male with renal cell carcinoma associated with erythrocytosis. Serum erythropoietin fluctuated with progression of the disease. Preoperative elevated erythropoietin (0.11 U/ml, p greater than 0.05) subsided after nephrectomy and again increased with developing lung metastasis (0.1 U/ml, p greater than 0.02). Erythropoietin was markedly increased in the tumorous extracts from primary renal cell carcinoma in the kidney (0.2 U/g, p greater than 0.01) and lung metastasis (0.8 U/g, p greater than 0.01). Renal cell carcinoma from the lung metastasis was transplanted into nude mice, resulting in erythrocytosis in some of these mic. In the erythrocytotic mice, erythropoietin was elevated to levels of 0.25--0.9 U/g (p greater than 0.01) in the tumorous extracts and increased (0.67 U/ml, p greater than 0.02) in the serum. These results indicate that this renal cell carcinoma is an erythropoietin-producing tumor, and this tumor has been successfully transplanted in nude mice for the first time.


Principles of blood separation and component extraction in a disposable continuous-flow single-stage channel. A single-stage disposable channel and seal that provides for leukocyte and granulocyte collection by continuous-flow cell separation (CFCS) has been designed by the IBM Corporation. This paper describes (1) the separation characteristics of whole blood as it responds to varying gravitational (G) forces and flow rates through the channel; (2) the mechanism by which the buffy coat accumulates and is extracted; (3) the efficiency of extraction; (4) those donor and procedural variables that contribute to the final yield; (5) posttransfusion increment response in patients; and (6) the functional integrity of the cells collected.


The storage of hard-packed red blood cells in citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1). The preservation of red cells "hard packed" to a hematocrit of over 80% from blood collected in citrate-phosphate-dextrose (CPD) or CPD-adenine (CPDA-1) has been investigated. After 21 days of storage, cells that had been collected in CPD solution had consumed most or all of the available glucose and manifested markedly impaired viability after reinfusion into the normal donor. In contrast, red cells prepared from blood collected in CPDA-1, a medium containing supplementary adenine and an increased amount of glucose, maintained higher glucose and adenosine triphosphate levels and, in most instances, manifested satisfactory posttransfusion viability. We emphasize that in addition to providing longer shelf life of stored blood, CPDA-1 provides a better hard-packed red cell concentrate for transfusion at 21 days.


Factor VII Padua 2: another factor VII abnormality with defective ox brain thromboplastin activation and a complex hereditary pattern. A new factor VII abnormality is presented. The propositus was a 9-yr-old child who presented a mild bleeding tendency characterized by epistaxis and easy bruising. The parents were not consanguineous, but they came from the same area. The laboratory features were mild prolongation of prothrombin time and P.P. test and normal partial thromboplastin and Stypven cephalin clotting times. The Thrombotest was moderately prolonged. Factor VII was 40%-50% of normal using rabbit or human brain thromboplastin, but only 13%-24% using ox brain thromboplastin. Factor VII cross-reacting material (CRM) was about 50% of normal. The father, a paternal aunt, and a paternal cousin showed similar clinical and laboratory findings. The brother of the propositus, the mother, and other members of her family showed about 50% factor VII activity and CRM and were considered to be heterozygotes for true factor VII deficiency. Similar findings were also present in the father and in the brother of the affected cousin. The defect in the propositus seems to consist of a double heterozygosity between abnormal factor VII and heterozygous factor VII true deficiency. The factor VII abnormality appears to consist of abnormal reactivity toward ox brain tissue thromboplastins and appears to be different from previously described factor VII abnormalities. The name factor VII Paudua2 is proposed for this condition.


Release of platelet fibronectin (cold-insoluble globulin) from alpha granules induced by thrombin or collagen; lack of requirement for plasma fibronectin in ADP-induced platelet aggregation. Platelets lysed with Triton X-100 contain 3.44 +/- 1.27 (SD) microgram of fibronectin (cold-insoluble globulin) per 10(9) platelets. Fibronectin was partially released from washed whole platelets by collagen or thrombin, and its release by collagen was inhibited by aspirin. Analysis of subcellular fractions obtained by density-gradient centrifugation of disrupted platelets indicated that fibronectin was contained in the alpha granules. Fibrinogen depleted of fibronectin (less than 2 microgram/mg) supported ADP-induced aggregation as effectively as fibrinogen contaminated with this protein, thus reinforcing the generally held view that fibrinogen itself is the necessary protein cofactor in this reaction.


Responses of the internal carotid artery to different endogenous vasoconstrictor substances. In order to study the action of serotonin (5-HT), noradrenaline (NA), hypertensin (HT), prostaglandins A1, B1 and E2 (PGA1, PGB1 and PGE2) and vasopressin (VP), internal carotid arteries were isolated in situ from both cerebral and general circulation and perfused continuously with oxygenated Ringers' bicarbonate solution. The order of potencies of the vasoactive substances when administered intra-arterially was: 5-HT greater than HT greater than PGE2- greater than PGB1 greater than NA. The relative duration of the constrictor effects was: 5-HT less than PGA1-less than HT and PGE2 less than PGB1 and NA less than VP. The relaxation index of these substances on the vascular wall was: 5-HT less than PGE2 less than HT less than PGB1-less than NA less than PGA1 less than VP. Some of these substances, specifically PGB1, PGE2 and VP, frequently caused a residual constriction of the smooth muscle following their dilator effect. The role of these vasoactive substances in the development of vasospasm is discussed.


[Relations between chromosomal findings and prognosis in acute nonlymphoblastic leukemia (author's transl)]. In 48 patients with acute non-lymphoblastic leukemia, all of whom had been treated according to the protocol 7421 of the "acute leukemia group B", remission rates and survival times were correlated with the chromosome constitution of bone marrow cells at diagnosis. 45.8% of the patients had only normal metaphases (N-patients), 31.3% had normal and abnormal metaphases (AN-patients), and 22.9% had only abnormal metaphases (AA-patients). Chromosomal findings were unrelated to patients' age. The remission rate of the N-patients was 72.7%, of the AN-patients 60%, and of the AA-patients 36.4%. The respective median survival times were 12.5, 8.5 and 4.0 months. The difference in remission rates and survival times between patients with normal and without normal metaphases was significant. Once a remission had been obtained the prognosis was similar among the 3 groups. The better prognosis of the AA-patients in this study as compared to previous reports might be related to a more effective chemotherapy.


Culture of human haemopoietic cells in diffusion chambers. I. Growth of normal peripheral blood cells. Growth of mononuclear cells from human peripheral blood from 10 normal individuals was tested in diffusion chamber culture over a period up to 17 or 21 days. After an initial decrease during the first few days an increase of the total cell number was observed with maximal values on day 13. In all individuals growth of undifferentiated blast cells, lymphocytes, plasma cells, immature and mature granulopoietic cells, macrophages, and megakaryocytes occurred. In all individuals the different cell types had similar growth patterns in diffusion chamber culture. The considerable numerical variations which were seen in the granulopoietic cells were probably due to different stem cell concentrations in the peripheral blood of the investigated individuals. The results indicate that the diffusion chamber technique is a valuable method for the detection of haemopoietic stem cells and the culture of lymphocytic cells in man.


Intestinal absorption of cobalt and iron: mode of interaction and subcellular distribution. 1. The absorption kinetic of 59Fe-(FeCl3) and 60CO-(CoCl2) 10 min after administration of increasing doses (0.5--1,000 nmoles metal) into tied-off duodenal segments of normal and iron-deficient rats shows saturation characteristic for both metals; in iron-deficient rats the absorption of both metals was enhanced. 2. The addition of increasing amounts of cobalt to the 59Fe-containing test solutions caused a decrease of the absorption of iron. 3. The study of the time dependence of this interaction in iron-deficient rats revealed, that cobalt inhibits the release of iron from mucosal cells into the blood, whereas the uptake of iron from the lumen into the mucosal cells did not differ from the controls without administration of cobalt. 4. The subcellular distribution of 59Fe and 60 Co in mucosal cell homogenates of iron-deficient rats after ultracentrifugation on a polyvinylpyrrolidone-CsCl solution shows a similar pattern for both metals; in the presence of cobalt the subcellular distribution of 59Fe is not changed. 5. From these results the conclusion is drawn that cobalt inhibits iron absorption not by an interference with iron binding sites on or in the luminal membranes of the mucosal cells but by an interaction with the releasing process at the contraluminal side.


Polycythemia vera: electron microscopy of the bone marrow in 10 non-treated patients. A thin section and freeze-fracture study. Electron microscopy (thin sections and freeze-fracture replicas) was performed on the bone marrow of ten patients with Polycythemia vera prior to any treatment. In addition to a hyperplasia of all three cell lineages and the sinuses, atypias were observed in the maturing erythroblasts. These aberrations of normal development consisted mainly of deep invaginations of the nuclear envelope in proerythroblasts and conspicuous nuclear clefts in erythro- and normoblasts. In comparison with similar changes in dyserythropoietic and aplastic anemia as well as leukemia these alterations are discussed in connection with disturbances of DNA synthesis. Further atypias involved megakaryopoiesis which displayed microforms probably as an evidence for maturation arrest. These ultrastructural abnormalities and their morphological features of a neoplastic proliferation of all three cell lineages in Polycythemia vera are in good agreement with the new concept of a transformation of a pluripotent stem cell with clonal character.


[Circulating immune complexes of patients with malignant lymphomas (author's transl)]. Using the 125J-Clq deviation method according to Sobel, the polyethylenglycol precipitation test of Nydegger, and a Sepharose 6B column chromatography in a modification of MacLennan's method, circulating immune complexes (CIC) have been detected in 20--27% of patients with Hodgkin's lymphoma, in 8% of patients with non-Hodgkin lymphoma of low, and in 20--40% with high malignancy. CIC appeared in advanced stages of the diseases and correlated well with the activity of the disease. CIC disappeared in remissions and reappeared during reactivation of the disease. CIC were only found in patients with general symptoms, i.e. B-symptoms. The pathogenetic role of CIC in the initiation of B-symptoms and in tumor spread is still not fully explained. The antibody part of the CIC is IgG, the antigenic component has not yet been characterized.