The isolation of myxomyosin,an ATP-sensitive protein from the plasmodium of a myxomycete

1. A procedure has been developed for the preparation of an active concentrate from the myxomycete, Physarum polycephalum. This concentrate responds with a lowered viscosity to the addition of small amounts of ATP. The preparation recovers in viscosity, and the process may be repeated. 2. In the most active concentrates, 75 per cent of the non-dialyzable material moves as a single boundary both in the descending limb in electrophoresis and in the ultracentrifuge. It contains about 10 per cent ribonucleic acid, which is at least in part reversibly bound to the protein. 3. The active material has been designated myxomyosin because of its origin and its similarity to actomyosin in ATP response.     

二株固氮芽孢杆菌的固氮特性研究

在无氮Ｈｉｎｏ培养基ＪＲ_１菌株的乙炔还原活性在氧浓度３％时最高,ＺＺ_１２菌株则随氧浓度上升而下降,但两菌株在空气条件下也能有效固氮;在有０．０１％酵母膏存在时,各氧气浓度菌株的乙炔还原值起伏不大。０．０２％铵盐存在时ＪＲ_１乙炔还原作用最强,但活性随硝酸盐浓度上升而一直下降;０．０２％硝酸盐时ＺＺ_１２菌株乙炔还原力最强,但其活性值随铵盐浓度递增而逐减。土壤中存在可利用糖类时ＪＲ_１和ＺＺ_１２     

Preservation of red cell concentrates. Dependence on storage time of IgG binding, osmotic fragility, MCV, and surface area index

In a comparative study of red cells from concentrates preserved in SAG medium without and with 30 mM sucrose, mannitol or sorbitol, resp., we determined the variation of IgG binding, osmotic fragility, MCV and the surface area index with storage time. IgG binding gave no conclusive results. Osmotic fragility turned out to be increased in simple SAG in comparison to the sugar-supplemented media. From measurements of microhematocrit and pH, the mean cellular volume (MCV), standardized to the initial pH value, turned out to decrease in all the media tested by not more than about 5 per cent after 3 weeks, and 10 per cent after 6 weeks. This is in advantageous contrast to the strong decrease in microhematocrit formerly observed in red cell concentrates in CDS-AG medium. Cells resuspended in simple SAG medium exhibited the smallest decrease in MCV. However, as inferred from data on hemolysis and vesiculation (D. Stibenz, accompanying paper), in these cells the loss of surface area proved to be maximal.     

Isolation of Actinomycetales from the soil of Kazakhstan on selective media with antibiotics

About 3000 actinomycetes were isolated from various soil samples collected in 11 regions of Kazakhstan. 62.7 per cent of them proved to be antagonists. For isolation of the strains, selective media supplemented with antibiotics were used. Kanamycin promoted growth of Actinomadura and Nocardia. Rubomycin promoted growth of Actinomadura. Tavromycetin and roseofungin were used as selective agents for the first time. Tavromycetin favoured isolation of Actinomadura and Nocardia. Roseofungin favoured isolation of Actinomadura. Light chestnut and serozemic soils were the most rich in antagonists (67.1 and 61.3 per cent, respectively) while saline and chestnut soils were the poorest in antagonists (32.2 and 30.6 per cent, respectively). Actinomadura were more frequent in light-chestnut light-loamy and serozemic soils. Half of the antibiotics isolated in the form of concentrates were identified with the known antibiotics or classified as belonging to various groups. A culture producing a novel antibiotic was isolated.     

THE USE OF PHOSPHOLIPASE C IN THE STUDY OF RECEPTORS FOR NEUROMUSCULAR BLOCKING AGENTS

Abstract— Segments of diaphragmatic muscle and cerebral nerve-ending particles from the rat were treated exhaustively (180 min) with high concentrations of phospholipase C (300 μg/ml) (EC 3.1.4.3). This enzyme caused loss of approx. 50 per cent of muscle membrane P and 70 per cent of nerve-ending membrane P. At lower concentrations (3 μg/ml; 180 min), phospholipase C released 25 per cent muscle membrane P and 55 per cent of nerve-ending membrane P. At the lower concentration (3 μg/ml; 180 min), the enzyme caused a slow but progressive loss (90 per cent) of muscle twitch amplitude evoked by nerve stimulation (phrenic nerve-hemidiaphragm preparation). Loss of muscle twitch amplitude was notably slowed by removal of the enzyme. Phospholipase C-treated neuromuscular junctions developed no resistance to hemicholinium, botulinum toxin, or d-tubocurarine. Such preparations were unusually sensitive to the neuromuscular blocking action of di-isopropylfluorophosphate. The data do not encourage a belief that phospholipids which are substrates for phospholipase C are receptors for hemicholinium, botulinum toxin, d-tubocurarine or di-isopropylfluorophosphate.     

Double-stranded RNA from phage F6: its interferon-inducing and antiviral properties]

Double stranded RNA was isolated from bacteriophage phi 6 parasitizing on phytobacteria. Its interferon-inducing and antiviral activities were shown in vitro and in vivo. In the culture of L-929 cells, interferon resistant to heat and acids was synthesized. The interferon could be practically completely neutralized by specific anti-interferon serum. The phage phi 6 preparation dsRNA in the lyophilized form was studied. The preparation retained its biological activity. It was shown that a preparation containing 30 per cent of dsRNA was less toxic than a preparation containing 100 per cent of dsRNA, the difference in the interferon-inducing activity being insignificant.     

Cytogenetic findings in acute myelogenous leukemias (FAB M 1 to M 6)

The results published in the period from 1973 to 1983 entitled "Cytogenetic findings in acute myeloic leukemias" (M 1 to M 6 of FAB classification) were compiled. In 50-60 per cent of those patients affected with acute myeloic leukemia a deviating karyotype could be detected. With a markedly higher frequency chromosomes 8 and 21 will take part in aberrations, with translocations (8; 21) having the main share with about 30-40 per cent. More than half the male bearers of translocation exhibits a loss of the Y-chromosome, a third of female patients a loss of the X-chromosome. Trisomy 8 and 9 as well as monosomy 7 appear in about 20 per cent. These aberrations can also be found in all other leukemic and preleukemic processes. Patients with karyotypic abnormalities in all their cells will have the slightest average survival time and the worst appeal to therapy. The sole appearance of monosomy 7 or Ph1-chromosome respectively seems to be an unfavourable sign from a prognostic point of view. Children with acute myeloic leukemia will possess an aberrant karyotype more frequently than adults, but they have a longer average life, boys are more frequently affected by this. Acute promyelocytic leukemia can be characterized cytogenetically in 94 per cent of the cases by translocation (15; 17). However, distinct geographical differences can be observed here, the causes of which have not been elucidated. About 40 per cent of the patients with acute myelo-monocytic leukemia developed aberrations. Further investigations will have to show whether the chromosome 11 really took part in it somewhat more frequently than merely at random. Chromosome anomalies have not a visible influence on the course of the disease. In 30-40 per cent of patients with a rarely occurring acute monocytic leukemia, an abnormal karyotype could be found. There was an incidence of 47 per cent for a specific translocation (9; 11) or a similar variant respectively. Erythroleukemia is characterized by a high instability of chromosomes and karyotypical variability, particularly in erythrocyte precursors and by an average survival time of one months. Megakaryoblastic and eosinophilic leukemia are very rare kinds of acute leukemias. The small number of publications allows no general statement to be made concerning karyotypical changes.     

Stability and optimization of canalicular function in hepatocyte couplets

An enriched preparation of rat hepatocyte couplets was obtaied by collagense perfusion and subsequent elutriation (> 85 per cent couplets and triplets; viability of over 95 per cent). Canalicular secretory activity (the ability to accumulate cholyl-lysyl-fluorescein, CLF) was first apparent after 2 h of culture at 37°C and was present in over 80 per cent of the total population after 5–6 h. This remained almost constant for at least 4 h in both elutriated and directly plated cells. Initial storage of freshly prepared couplets at 4°C for up to 6 h prior to incubation had no adverse effect upon secretory function. Reduction of canalicular secretory activity occurred at a concentration of the hepatotoxic agent menadione (IC₅₀ 17 μM) that was lower than that required to induce mild plasma-membrane blebbing (IC₅₀ 43 μM). This study has optimized and characterized the canalicular secretory effectiveness and stability of an enriched preparation of hepatocyte couplets, and established the feasibility of studies of toxic agents on hepatobiliary function in a heterogeneous population of hepatocytes. In this preparation other biochemical parameters can be assessed, thus complementing previous techniques using individual couplets.     

Cytochemical studies of mitochondria. II. Enzymes associated with a mitochondrial membrane fraction

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained approximately 12 per cent of the protein N and approximately 35 per cent of the phospholipide of the whole mitochondria and accounted for approximately 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.     

THE TREATMENT OF METASTATIC BREAST CANCER IN BONE

The author report clinical experience with 212 cases of mammary cancer metastatic to bone, in 186 of which radiotherapy was given, and in 26 steroid hormone therapy.At least 70 per cent of patients with bone metastasis from breast cancer were relieved of pain by adequate roentgen therapy, the relief lasting for most of the survival time in many instances.About 25 per cent of patients had recalcification or reossification of bony lesions with roentgen therapy; while dramatic, this is not always an indication that relief of pain will continue or that survival time will be lengthened.If and when adequate radiotherapy has not been effective or cannot be administered (for example, in a patient with extremely widespread metastasis, or one residing at a considerable distance from radiotherapeutic service) steroid hormone therapy in adequate dosage is frequently beneficial. From 40 per cent to 75 per cent of patients with bone metastases from breast cancer are relieved of pain by steroid hormone therapy. In about 15 per cent of cases recalcification of the lesion occurs.Effective roentgen therapy may usually be given in a relatively brief period of time (one to two weeks). Effective steroid hormone therapy usually requires from 12 to 24 weeks.Complications of steroid hormone therapy are numerous. Some patients are made considerably worse by such therapy. These complications may only be controlled by reduction or discontinuation of the hormones. For this reason, it is recommended that irradiation always be used as the initial method of palliation.     

Influence des lecithines dicarboxyliques sur les liposomes

We have investigated the effect of dicarboxylic lecithins on liposomes prepared from egg-lecithins and their incorporation into liposome preparation.In the presence of phospholipids, liposome cell permeability to glucose is five times higher than in controls.The incorporation of dicarboxylic lecithins into liposomes at a molar concentration of 50 molecules per cent reduces by 60% the amount of glucose trapped inside the liposomes.Measurements of ovelecithin-glutaryl-lecithin liposomes stability show that stable structures are formed at a molar concentration of 70 molecules per cent in the presence of cholesterol. On the contrary, the liposomes prepared in the absence of cholesterol are not stable at a molar concentration of 50 molecules per cent.Measurements of sedimentation of mixed dispersions of monocarboxylic lecithins and glutaryl-lecithins show that glutaryl-lecithins must be in the form of micelles to solubilize the phosphatidyl cholines.     

Improving the quality of washed and buffy coat-free erythrocyte concentrates]

Washing buffy-coat free erythrocyte concentrates three times in bottles used for blood storage will diminish their leukocyte content to 0.22 +/- 0.11 x 10(9) per TE (= 9% of the initial value in whole blood, and the thrombocyte content to 0.3 +/- 0.5 x 10(9) per day (= 2% of the initial value in whole blood). Even 50% of leukocytes (mainly lymphocytes) and 80% of thrombocytes are eliminated simply by buffy coat separation. 30% of erythrocytes are lost by the washing process. Due to increasing haemolysis (0.22%) a subsequent storage of 24 hours should not be exceeded for washed erythrocyte concentrates. Further quality parameters, such as morphological index, pH, ATP, 2,3-P2G and K+ and Na+, were investigated. As far as selected quality parameters are concerned, washing erythrocyte concentrates three times in bottles for blood storage may be compared with washing them once in blood bags. The present findings confirm the conclusion that the washing of erythrocyte concentrates with a solution of sodium chloride in order to eliminate leukocytes may for the most part exclude non-haemolytic febrile transfusion reactions, but not immunization. More effective procedures of eliminating leukocytes, such as filtration, TTK or even glycerin, treatment of erythrocyte concentrates without cryoconservation, are indispensable.     

CYTOCHEMICAL STUDIES OF MITOCHONDRIA : II. ENZYMES ASSOCIATED WITH A MITOCHONDRIAL MEMBRANE FRACTION

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ~ 12 per cent of the protein N and ~ 35 per cent of the phospholipide of the whole mitochondria and accounted for ~ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.     

Correlation between [U-14C]glucose metabolism and function in perfused cat brain

In brain perfusion experiments conducted with blood containing [U-14C]glucose the relative specific activity (RSA) of blood glucose carbon incorporated in brain intermediate metabolites was measured. It was demonstrated that the so-called metabolic pattern of Geiger is not constant, but it bears a close relation to the function of the brain. The results of the study may be summarized briefly as follows. (1) In a group (A) of cats with a high level of brain function, the RSA of lactic acid was 75 per cent; that of glutamic acid 80 per cent; aspartic acid 75 per cent; glutamine 61 per cent; GABA 43 per cent; and respiratory CO2 55 per cent. It was observed that the major part of the carbon of amino acids, such as glutamic acid and aspartic acid, which are directly associated with the tricarboxylic acid cycle are derived from blood glucose. (2) In a group (B) showing a low level of brain function, the RSA of each amino acid was considerably lowered. The RSA of glutamic acid and aspartic acid was about 50 per cent and that of respiratory CO2 was 27 per cent. (3) In a group (C) with a still lower level of brain function, each amino acid as well as the respiratory CO2 had still lower RSA values. (4) The metabolic pattern of Geiger corresponds to values obtained during low functional activity of the brain in our experiment.     

Antibacterial and therapeutic effectiveness of a pectin from sea grass Zostera

The kinetics of the antibacterial activity of zosterin, a polysaccharide preparation of a sea grass belonging to Zoster, was studied. By its chemical structure zosterin is a low ++methoxylated pectin. In vitro the preparation markedly inhibited the growth of ++Gram-negative and ++Gram-positive organisms: S. aureus, E. coli, Y. pseudotuberculosis, S. typhimurium and Ps. aeruginosa. On a model of experimental pseudotuberculosis++ infection caused by oral contamination of mice F1 (CBA X C57B1) with a suspension of Y. pseudotuberculosis zosterin was shown to have a therapeutic effect. It protected 30 to 40 per cent of the animals when administered per os simultaneously with or 24 hours after the contamination. The results are in favour of the zosterin further investigation as a preparation useful in prevention of intestinal infections in persons being in contact with the patients.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

Continuous,high capacity reconstitution of nutrient media from concentrated intermediates

We designed an Integrated Media Preparation System (IMPS) for continuous, on-line preparation of cell culture media and delivery to intermediate storage vessels or directly to a bioreactor. Key components of the IMPS include: a high precision, continuous fluid mixing device; formulation-specific liquid medium concentrates; validated process controls and membrane filtration; and automated dispensing into large volume flexible plastic containers. The IMPS system is designed to produce sterile, single-strength liquid medium from common raw materials at a delivery rate of 1000–3000 liters per hour and will manufacture homogenous batches from several thousand liters to over 60,000 liters. Fortified nutrient media prepared from multi-component 50X concentrates have been demonstrated to accelerate bioreactor seed chains, increase product yield, and reduce the overall manufacturing cost of nutrient medium. A productivity matrix will analyze the fully-loaded costs and contrast alternative methods for media preparation against projected biological yield.Abbreviations IMPS Integrated Media Preparation System - 50X Nutrient fluid components formulated at fifty-fold final use concentration - 1X Nutrient fluid formulated at final, single-strength use concentration - cGMP Current Good Manufacturing Practices - SCADA Supervisory Control and Data Acquisition - PLC Process Logic Controller - LTI Life Technologies, Inc. - WFI Water for Injection - CIP Clean in place - SIP Sterilize in place - HPLC High performance liquid chromatography - DMEM Dulbecco's Modified Eagle's Medium     

AN ELECTRON MICROSCOPE STUDY OF CALF THYMUS NUCLEAR PREPARATIONS ISOLATED IN SUCROSE SOLUTIONS

This work was carried out with the intent of developing a method capable of routinely evaluating calf thymus nuclear preparations with the electron microscope. Examination of small random samples, pre-embedded in agar after fixation with permanganate, were found to give results comparable to those obtained with much larger samples withdrawn randomly from pellets and embedded and sectioned conventionally. Results obtained by this pre-embedding technique with acrolein, osmium tetroxide, or permanganate fixations were equivalent. Calf thymus nuclear preparations isolated in sucrose by the method prevalently used (see 1) are contaminated only slightly with intact cells, to a degree which varies with each preparation. However, intact cells, damaged cells, or nuclei with some cytoplasm constitute together about 30 per cent of the preparation. Particles other than intact cells are not readily distinguishable from one another by light or phase microscope techniques. These preparations can be purified further by centrifuging through a dense sucrose layer. In our hands, however, contamination with some cytoplasm still remains in approximately 10 per cent of the particles. Incubation of the particles prepared without purification procedures, under conditions frequently used, results in the extensive breakdown of particles. Under at least one set of conditions, nuclei are selectively disrupted, leaving primarily damaged cells in the preparation.     

MICROSOMAL NUCLEOPROTEIN PARTICLES FROM PEA SEEDLINGS

Ultracentrifugal analysis of an extract of pea epicotyls, previously freed of debris and larger particles by centrifugation at 40,000 g for 10 minutes, has revealed the presence of a major component which possesses a sedimentation coefficient of 74 S. This component constitutes about 25 per cent of the TCA-precipitable material in the clarified epicotyl extract and is estimated to make up 1 to 2 per cent of the dry weight of the original tissue. In size, chemical composition, and morphology, the 74 S component resembles the nucleoproteins of the microsomes from animal tissues. The 74 S component of pea epicotyl extracts has been purified by repeated cycles of differential centrifugation to yield a preparation which is 80 per cent homogeneous in the analytical ultracentrifuge. It has been found to contain 30 to 37 per cent RNA as judged by a variety of analytical techniques. Approximately 55 per cent of the weight of the material is protein and a further 4.5 per cent phospholipide. Electron micrographs of air-dried specimens of the purified preparation show the 74 S constituent to be flattened spheres with an average height of 180 A and an average diameter of approximately 280 A. The molecular weight of the 74 S particles is computed from sedimentation, viscosity, and partial specific volume data to be 4.5 million ± 10 per cent in agreement with the value estimated from electron micrographs. The 74 S or microsomal component of pea epicotyls is rapidly aggregated in the presence of low concentrations of Mg ions or by somewhat higher concentrations of Ca or K salts. ATP on the contrary causes resolution of electrolyte-induced microsomal aggregates with simultaneous degradation of the particles to an ultracentrifugally inhomogeneous mixture of lower molecular weight materials.     

The value of the free hydroxyproline and the N-acetyl-beta- glucosaminidase for the observation of the course of malignant tumors

In 172 patients with various malign tumours the free hydroxyprolin (HP) and N-acetyl-beta-glucosaminidase (beta-NAG) in the serum were determined. The established values were analysed according to the kind of tumour, spread of tumour, behaviour of growth and histological classification and statistically evaluated. In mamma carcinoma progressively metastasing into bones HP is significantly increased in its mean value. 71.4 per cent of the patients have pathological values. The parameter for bone-marrow diagnostics failed in 28.6 per cent. Increases may only occur until 9 weeks before traditional diagnostics of metastases. beta-NAG values lie within the normal range for clinically not metastasing mamma carcinoma. The enzyme level will increase significantly after each tumour progression into an organ. The diagnostic sensitivity for the general diagnostics of metastases amounts to 97.7 per cent. Increases without any evidence of metastases are either due to other diseases with connective tissue changes or to occult micrometastases. An exact control is required for these patients. Likewise, HP and beta-NAG have a prognostic importance for malign testicle tumours from nonseminomes character and malign lymphomas, because with tumour progression they will turn into pathological ones. Cytostatics have no direct impact on the serum level of HP and beta-NAG.