NON-SUPPRESSIBLE INSULIN-LIKE ACTIVITY AND SULPHATION ACTIVITY IN SERUM EXTRACTS OF NORMAL SUBJECTS, ACROMEGALICS AND PITUITARY DWARFS

1976 ◽  
Vol 81 (1) ◽  
pp. 28-42 ◽  
Author(s):  
U. Schlumpf ◽  
R. Heimann ◽  
J. Zapf ◽  
E. R. Froesch
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Acetic Acid ◽  
Large Scale ◽  
Normal Subjects ◽  
Step Procedure ◽  
Chromatographic Procedure ◽  
One Step ◽  
Insulin Like Activity

ABSTRACT Non-suppressible insulin-like activity (NSILA) is a term used for a variety of substances in serum, excluding insulin, which promote glucose uptake of adipose tissue and diaphragm in vitro. NSILA-S is a peptide with a molecular weight of 7000 which is soluble in acid ethanol and which has been purified on a large scale from human serum. This study describes a simple chromatographic one step procedure by which NSILA-S can be extracted and quantitatively measured in individual sera. Using Sephadex G-75 equilibrated with 1 m acetic acid, NSILA-S was detected only in one peak containing small molecular peptides. NSILA-S obtained with this one step chromatographic procedure exerted all the effects of purified NSILA-S including sulphation activity on the rat cartilage. All chromatographic fractions with NSILA-S also had sulphation activity. Both, NSILA-S and sulphation activity were increased in acromegalics and decreased in pituitary dwarfs. It is suggested that one molecule in serum is responsible for both activities which are, at least in part, under the control of growth hormone.

scholarly journals An effective GC method for the determination of the fatty acid composition in silkworm pupae oil using a two-step methylation process

2015 ◽  
Vol 80 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Jun Wang ◽  
Weiwei Wu ◽  
Xudong Wang ◽  
Min Wang ◽  
Fuan Wu
Keyword(s):  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Acid Composition ◽  
Methyl Esters ◽  
Superior Performance ◽  
Step Procedure ◽  
Silkworm Pupae ◽  
Chromatographic Procedure ◽  
One Step ◽  
Synthesis Yield

In search of an accurate and effective method to determine fatty acid composition in silkworm pupae oils, five methylation methods were evaluated for use in the gas chromatographic (GC) quantitation of fatty acid methyl esters (FAMEs), including one-step esterification catalyzed by an acidic (H2SO4 and BF3) or alkali catalyst (KOH and NaOCH3) and a two-step procedure catalyzed successively by KOH and H2SO4. These methods were comparatively adopted to quantify FAMEs in silkworm pupae oil using GC-MS and GC and then validate their precision, stability and average recovery rates. The results indicated that compared with the other four methyl esterification methods, two-step methylation effectively improves the synthesis yield of FAMEs, conserves agents, and eliminates the usage of potential harmful reagents. The proposed GC method has been validated, shows good accuracy and precision, and has been applied successfully to the quantification of FAMEs in several varieties of silkworm pupae oils. The short analytical run time leads to low costs and a fast chromatographic procedure. In summary, two-step pretreatment had superior performance, providing technical references for the determination and analysis of fatty acids in other oils.

Steroid and bile acids amide conjugates with D-glucosamine

2011 ◽  
Vol 76 (1) ◽  
pp. 65-74
Author(s):  
Zdena Nováková ◽  
Jana Tomanová ◽  
Lucie Štěrbová ◽  
Pavel Drašar
Keyword(s):  
Acetic Acid ◽  
Bile Acids ◽  
Acid Chloride ◽  
Acetyl Chloride ◽  
Glucosamine Hydrochloride ◽  
Negative Results ◽  
Step Procedure ◽  
One Step ◽  
New Type

New type of amide conjugates of steroid and bile acids with D-glucosamine 1 and 2 were prepared. Title compounds are prepared via acid chloride or using N-[({[(1E)-1-cyano-2-ethoxy-2-oxoethylidene]amino}oxy)(dimethylamino)methylidene]-N-methylmethanaminium tetrafluoroborate as condensation agent. They were examined for gelation properties with negative results. Per-O-acetylated D-glucosamine hydrochloride was prepared in one step procedure from D-glucosamine hydrochloride by acetylation in a mixture of acetyl chloride and acetic acid.

IMMUNOREACTIVE GROWTH HORMONE IN HUMAN URINE

1972 ◽  
Vol 71 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Kristian F. Hanssen
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Linear Relationship ◽  
Human Urine ◽  
Gel Filtration ◽  
Human Growth ◽  
Normal Subjects ◽  
Normal Human ◽  
Radio Immunoassay

ABSTRACT By using a double antibody radio-immunoassay (pre-precipitation technique) for the determination of immunoreactive human growth hormone (IRHGH) in normal human urine concentrated by dialysis and lyophilization, a factor was revealed that displaces 125I-HGH from HGH antibodies. This displacement was neither due to salts nor to glucose; it is suggested that it is due to IRHGH in the urine. A linear relationship between dilution of urine and the measured IRHGH concentration was obtained. Recovery of exogenous HGH was between 70–105%. The recovery of IRHGH from different volumes of urine following dialysis and lyophilization was between 97–110%. Plasma IRHGH and urinary IRHGH was measured simultaneously after HGH injection in a normal subject. A correlation was shown between plasma IRHGH and urinary IRHGH. In 9 normal subjects, the urinary IRHGH ranged from 28–53 ng/24 h. The excretion of urinary IRHGH was increased in acromegaly and was diminished in some, but not in all patients with adult hypopituitarism. The urinary IRHGH was further studied by gel filtration. It was recovered in one peak corresponding to a molecular weight of approximately 20 000 – 30 000. However, in the present work it was not clarified whether the urinary IRHGH represents pituitary HGH excreted in the urine or a metabolite of high molecular weight with retained immunological properties.

Implantable insulin pumps: diagnosis and management of decreased flow rates using radionuclide investigation

1996 ◽  
Vol 19 (12) ◽  
pp. 723-729
Author(s):  
H. Boulahdour ◽  
A. Behar ◽  
M.-J. Haardt ◽  
J-L. Selam
Keyword(s):  
Insulin Pumps ◽  
Distal Catheter ◽  
Non Invasive ◽  
Step Procedure ◽  
Catheter Obstruction ◽  
Catheter Malfunction ◽  
Invasive Method ◽  
One Step ◽  
Isotonic Sodium Chloride

The aim of this study was to develop a diagnostic procedure for pumping unit malfunction by radionuclide imaging (RI) and to validate the method by comparing the results with those obtained using more conventional methods. Fifteen radionuclide investigations were performed in 11 patients with intraperitoneal implantable insulin pumps. One mCi of 99 mTc in 1 ml isotonic sodium chloride was injected into the reservoir. The results based on catheter visualization and peritoneal accumulation were compared blindly to the efficacy of alkaline rinses and laparoscopic findings. In all RI stoppage cases except one alkaline rinses failed to restore flow. Where laparoscopy was performed, comparisons were concordant i.e. no outflow from the tip of the catheter. The RI images obtained were reproduced in vitro using a pump under normal flow conditions and complete proximal and distal catheter obstruction. RI is a safe, quick non invasive method which allows the location of the site of pump/catheter malfunction within a one step procedure and the prediction of the efficacy of sodium hydroxide rinses.

Embryo Rescue Culture Medium for Improved Synthesis of 4x Wheat × Rye Hybrids

1994 ◽  
Vol 42 (4) ◽  
pp. 493
Author(s):  
CH Balatero ◽  
NL Darvey
Keyword(s):  
Embryo Rescue ◽  
Favourable Effect ◽  
Embryo Formation ◽  
Step Procedure ◽  
Hybrid Embryo ◽  
One Step ◽  
Well Differentiated ◽  
Semi Solid ◽  
Optimum Recovery

The cross-incompatibility barrier between 4x wheat and rye has limited the genetic base for triticale breeding. Experiments designed to improve the synthesis of wheat-rye amphihaploids were conducted. The effects of 2,4-D on crossability and 3x hybrid embryo differentiation, and the influence of one-step and two-step media on the culture of immature 3x embryos in vitro, were investigated. Application of 10 mg L-1 2,4-D slightly improved seed set but significantly reduced the frequency of normal embryos. In contrast to the reported favourable effect of 2,4-D on haploid embryo formation in wheat × maize crosses, the application of 2,4-D in the present study offers no real advantage on amphihaploid embryo formation from 4x wheat × rye crosses. For small and immature wheat-rye hybrid (3x) embryos, optimum recovery in vitro was obtained via a two-step procedure consisting of a semi-solid MN medium followed by MS medium supplemented with IAA (1 mg L-1) and BAP (1 mg L-1). For bigger and well-differentiated embryos, the use of a one-step Gamborg's B5 medium was sufficient.

Monoclonal antibodies reveal circulating growth hormone of high molecular weight not detectable by conventional assays

1990 ◽  
Vol 123 (3) ◽  
pp. 317-325 ◽  
Author(s):  
Marguerite Luthman ◽  
Ingileif Jónsdóttir ◽  
Bo Skoog ◽  
Inga-Lena Wivall ◽  
Paul Roos ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
High Molecular Weight ◽  
Specific Binding ◽  
Affinity Purification ◽  
Normal Subjects ◽  
Polyclonal Antiserum ◽  
Placental Lactogen ◽  
Gh Secretion ◽  
High Molecular Weight Proteins

Abstract. A radioimmunoassay based on a monoclonal antibody, Mc-ab 1, which was raised against growth hormone but cross-reacted with human placental lactogen yielded higher GH immunoreactivity levels in serum than one based on a polyclonal antiserum. This discrepancy was noted in subjects with normal GH secretion as well as in patients with GH insufficiency. To characterize this GH immunoreactivity detected by Mc-ab 1, affinity purification and molecular sieve chromatography of serum were performed. High molecular weight proteins with GH immunoreactivity were found with both techniques. These proteins were associated with carbohydrates. Affinity cross-linking showed specific binding of radiolabelled GH to high molecular weight proteins in the serum. After fractionation of serum, the GH immunoreactivity became detectable by the polyclonal antiserum assay as well as by an immunoradiometric assay. GH immunoreactive material with an approximate mass of 80 kD was subjected to isoelectric focusing. When GH immunoreactive fractions at pH 5 were re-chromatographed, GH immunoreactivity was recovered in the elution volume corresponding to monomeric GH. Our results show that sera from normal subjects as well as from patients with deficient GH secretion contain notable amounts of high molecular weight GH which is undetectable by antibodies generally used for GH measurements, but which can be revealed after fractionation of serum.

scholarly journals Mass Generation, Neuron Labeling, and 3D Imaging of Minibrains

2021 ◽  
Vol 8 ◽  
Author(s):  
Subashika Govindan ◽  
Laura Batti ◽  
Samira F. Osterop ◽  
Luc Stoppini ◽  
Adrien Roux
Keyword(s):  
Large Scale ◽  
3D Imaging ◽  
Cell Types ◽  
Mixed Population ◽  
Projection Neurons ◽  
Neuron Morphology ◽  
Mass Generation ◽  
One Step ◽  
Human Ipsc

Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains.

scholarly journals Biosynthesis And Structural Characterization of Levan By A Recombinant Levansucrase From Bacillus Subtilis ZW019

2021 ◽  
Author(s):  
Jingyue Wang ◽  
Xinan Xu ◽  
Fangkun Zhao ◽  
Nan Yin ◽  
Zhijiang Zhou ◽  
...  
Keyword(s):  
Large Scale ◽  
Fermentation Broth ◽  
Monosaccharide Composition ◽  
Enzymatic Reactions ◽  
Scale Production ◽  
Large Scale Production ◽  
Nmr Techniques ◽  
One Step ◽  
Activity Of Enzymes

Abstract Purpose: The yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan. Methods: A recombinant plasmid pET-28a-AcmA-Z constructed in the previous study was used to produce levansucrase. The effects of temperature, pH, and metal ions on the levan formation activity of the levansucrase were investigated. The polymer was analyzed by means of HPIC, FTIR, NMR techniques.Results: The recombinant levansucrase could be easily purified in one step and the purified enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions was optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K+ and Ca2+. The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was about 1.56×106 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2, 6) levan.Conclusions: The results of this study provide a basis for large-scale production of levan by enzymatic method.

In vitro synthesis of glycogen: the structure, properties, and physiological function of enzymatically-synthesized glycogen

Biologia ◽  
2011 ◽  
Vol 66 (3) ◽  
Author(s):  
Hideki Kajiura ◽  
Hiroki Takata ◽  
Tsunehisa Akiyama ◽  
Ryo Kakutani ◽  
Takashi Furuyashiki ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
Large Scale ◽  
Glycogen Synthesis ◽  
Average Molecular Weight ◽  
Spherical Particles ◽  
Average Chain Length ◽  
Scale Production ◽  
In Vitro Synthesis

AbstractThis review describes a new enzymatic method for in vitro glycogen synthesis and its structure and properties. In this method, short-chain amylose is used as the substrate for branching enzymes (BE, EC 2.4.1.18). Although a kidney bean BE and Bacillus cereus BE could not synthesize high-molecular weight glucan, BEs from 6 other bacterial sources produced enzymatically synthesized glycogen (ESG). The BE from Aquifex aeolicus was the most suitable for the production of glycogen with a weight-average molecular weight (M w) of 3,000–30,000 k. The molecular weight of the ESG is controllable by changing the concentration of the substrate amylose. Furthermore, the addition of amylomaltase (AM, EC 2.4.1.25) significantly enhanced the efficiency of this process, and the yield of ESG reached approximately 65%. Typical preparations of ESG obtained by this method were subjected to structural analyses. The average chain length, interior chain length, and exterior chain length of the ESGs were 8.2–11.6, 2.0–3.3, and 4.2–7.6, respectively. Transmission electron microscopy and intrinsic viscosity measurement showed that the ESG molecules formed spherical particles. Unlike starch, the ESGs were barely degraded by pullulanase. Solutions of ESG were opalescent (milky-white and slightly bluish), and gave a reddishbrown color on the addition of iodine. These analyses revealed that ESG shares similar molecular shapes and solution properties with natural-source glycogen. Moreover, ESG had macrophage-stimulating activity and its activity depends on the molecular weight of ESG. We successfully achieved large scale production of ESG. ESG could lead to new industrial applications, such as in the food, chemical, and pharmaceutical fields.

Neutrophil function in whole blood and after purification: Changes in receptor expression, oxidase activity and responsiveness to cytokines

1992 ◽  
Vol 12 (2) ◽  
pp. 123-133 ◽  
Author(s):  
Fiona Watson ◽  
John J. Robinson ◽  
Steven W. Edwards
Keyword(s):  
Whole Blood ◽  
Neutrophil Function ◽  
Receptor Expression ◽  
Complement Receptors ◽  
Step Procedure ◽  
Plasma Membrane Receptor ◽  
One Step ◽  
Reactive Oxidants ◽  
In Cells

Neutrophil function and plasma membrane receptor expression was measured in cell suspensions isolated by two separate procedures and in unfractionated whole blood. When cells were prepared by a combined dextran/ficoll procedure, their ability to generate reactive oxidants in response to fMet-Leu-Phe was greater than in corresponding cells isolated by a one-step procedure on Mono-Poly Resolving Medium (M-PRM). Cells prepared by both methods could be primed in vitro by rGM-CSF, but the priming ratio was greater in cells prepared by the latter method. The ability of neutrophils in whole blood to generate reactive oxidants in response to fMet-Leu-Phe was extremely low, but this was increased by more than 10 fold if the blood was pre-incubated with rGM-CSF. Similarly, expression of CD 11b and CD 16 was very low (or undetectable) in neutrophils in whole blood, but this was rapidly increased upon priming. Activation by PMA resulted in a down regulation of CD 16 expression as the receptor was shed from the cell surface. Neutrophils isolated by either the dextran/ficoll or the M-PRM method showed increased expression of receptors compared with those in whole blood, although this expression was lower in cells isolated by the latter method. These data indicate that the isolation procedures used to obtain purified neutrophils prime both receptor expression and oxidase function, although these effects are minimalised in isolation procedures using M-PRM. Furthermore, as CD 16 expression on neutrophils in whole blood is rapidly up-regulated during priming, it seems likely that, as for complement receptors, rapidly-mobilisable intracellular stores of this receptor exist.

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