NACM, a cytopathogen from Naegleria ameba: Purification, production of monoclonal antibody, and immunoreactive material in NACM-treated vertebrate cell cultures

1989 ◽  
Vol 93 (3) ◽  
pp. 391-401
Author(s):  
T. H. DUNNEBACKE ◽  
J. S. DIXON
Keyword(s):  
Mammalian Cells ◽  
Time Course ◽  
Large Mass ◽  
Biologically Active ◽  
Dot Blot ◽  
Free Living ◽  
Chromatographic Separations ◽  
Molecular Weight Range ◽  
Perinuclear Cytoplasm ◽  
Liquid Chromatographic

An amebic component NACM (Naegleria ameba cytopathogenic material) is obtained from free-living amebae and acts as a cytopathogen in cultured avian and mammalian cells. NACM has been purified and partially characterized using a scheme that includes ammonium sulfate precipitation, liquid chromatographic separations and electrophoretic isolations. The purified NACM product is biologically active. Its properties, revealed by its behavior following treatment with enzymes and during purification, are those of a protein; its size is in the molecular weight range of 36000 and it has an isoelectric point of pH4.2. Monoclonal antibodies have been produced to NACM that prevent its cytopathic activity, and, in dot-blot procedures, identify purified NACM-containing fractions. When used as immunostains, NACM antibodies disclose the presence of immunospecific material both in the amebae and in vertebrate cells inoculated with NACM. In the amebae, the immunostain is located at the tips of the pseudopodia and in the peripheral cytoplasm; in the vertebrate cells, it is absent in uninoculated cells and is not observed in the inoculated cells for the first 3–4 days after the addition of the NACM to the culture. Subsequently, in a time course associated with the development of cytopathic changes observed in the light microscope, immunostaining material develops in the perinuclear cytoplasm where it condenses into a large mass before the cells undergo lysis. On the basis of these results, it appears that NACM represents an unprecedented system in which a protein material from an organism, in the course of destroying unrelated cells, causes the production of a cytoplasmic product that is immunologically recognizable as the inoculating amebic material.

Eco-Friendly Green Liquid Chromatographic Separations of a Novel Combination of Azelastine and Fluticasone in the Presence of their Pharmaceutical Dosage form Additives

2020 ◽  
Vol 16 (3) ◽  
pp. 277-286
Author(s):  
Amal A. El-Masry ◽  
Mohammed E. A. Hammouda ◽  
Dalia R. El-Wasseef ◽  
Saadia M. El-Ashry
Keyword(s):  
Lower Limit ◽  
Dosage Form ◽  
Limit Of Detection ◽  
Sodium Dodecyl ◽  
Uv Detection ◽  
Simultaneous Estimation ◽  
Limit Of Quantification ◽  
Chromatographic Separations ◽  
Pharmaceutical Dosage Form ◽  
Liquid Chromatographic

Background: The first highly sensitive, rapid and specific green microemulsion liquid chromatographic (MELC) method was established for the simultaneous estimation of fluticasone propionate (FLU) and azelastine HCl (AZL) in the presence of their pharmaceutical dosage form additives (phenylethyl alcohol (PEA) and benzalkonium chloride (BNZ)). Methods: The separation was performed on a C18 column using (o/w) microemulsion as a mobile phase which contains 0.2 M sodium dodecyl sulphate (SDS) as surfactant, 10% butanol as cosurfactant, 1% n-octanol as internal phase and 0.3% triethylamine (TEA) adjusted at pH 6 by 0.02 M phosphoric acid; with UV detection at 220 nm and programmed with flow rate of 1 mL/min. Results: The validation characteristics e.g. linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), accuracy, precision, robustness and specificity were investigated. The proposed method showed linearity over the concentration range of (0.5-25 µg/mL) and (0.1-25 µg/mL) for FLU and AZL, respectively. Besides that, the method was adopted in a short chromatographic run with satisfactory resolution factors of (2.39, 3.78 and 6.74 between PEA/FLU, FLU/AZL and AZL/BNZ), respectively. The performed method was efficiently applied to pharmaceutical nasal spray with (mean recoveries ± SD) (99.80 ± 0.97) and (100.26 ± 0.96) for FLU and AZL, respectively. Conclusion: The suggested method was based on simultaneous determination of FLU and AZL in the presence of PEA and BNZ in pure form, laboratory synthetic mixture and its combined pharmaceutical dosage form using green MELC technique with UV detection. The proposed method appeared to be superior to the reported ones of being more sensitive and specific, as well as the separation was achieved with good performance in a relatively short analysis time (less than 7.5 min). Highly acceptable values of LOD and % RSD make this method superior to be used in quality control laboratories with of HPLC technique.

Macrophages produce somnogenic and pyrogenic muramyl peptides during digestion of staphylococci

1991 ◽  
Vol 260 (1) ◽  
pp. R126-R133 ◽  
Author(s):  
L. Johannsen ◽  
J. Wecke ◽  
F. Obal ◽  
J. M. Krueger
Keyword(s):  
Immune Response ◽  
Body Temperature ◽  
Bacterial Cell ◽  
Cell Walls ◽  
Mammalian Cells ◽  
Slow Wave Sleep ◽  
Biological Effects ◽  
Biologically Active ◽  
Chromatographic Behavior ◽  
Bacterial Cell Walls

Muramyl peptides have a variety of biological effects in mammals, including enhancement of the immune response, sleep, and body temperature. Although mammals lack biosynthetic pathways for muramyl peptides, they are found in mammals and are well known as components of bacterial cell walls. This suggests that phagocytic mammalian cells digest bacterial cell walls and produce biologically active muramyl peptides. Staphylococcal cell walls were radioactively labeled during growth of the bacteria. During the digestion of these radiolabeled bacteria, murine bone marrow macrophages produced low-molecular-weight substances that coeluted chromatographically with the radioactive cell wall marker. Further separation of these substances using reversed-phase high-performance liquid chromatography resulted in the isolation of substances with high specific biological activity. Intracerebroventricular injection of rabbits with these substances induced an increase in slow-wave sleep and body temperature and a suppression of rapid-eye-movement sleep. The characteristics of the biological responses and the chromatographic behavior of the active components are consistent with those of muramyl peptides. The ability of macrophages to tailor muramyl peptides from peptidoglycan may provide an amplification step for the immune response. Muramyl peptides released by macrophages may also act as mediators for various facets of the acute phase response elicited by bacterial infections such as fever and sleep.

Time-Course Studies of Phytoalexins and Glucosinolates in UV-Irradiated Turnip Tissue

1991 ◽  
Vol 46 (3-4) ◽  
pp. 189-193 ◽  
Author(s):  
Kenji Monde ◽  
Mitsuo Takasugi ◽  
Jenny A. Lewis ◽  
G. Roger Fenwick
Keyword(s):  
High Performance ◽  
Time Course ◽  
Brassica Campestris ◽  
Structural Features ◽  
Biologically Active ◽  
Indole Glucosinolates ◽  
Freeze Dried ◽  
Indole Phytoalexins ◽  
The Individual ◽  
Complex Manner

Sliced turnip root (Brassica campestris L. ssp rapa) was irradiated for a total of 20 min with a 15 W germicidal lamp and the tissue incubated at 25 °C. The effects of such treatment on indole phytoalexins (methoxybrassinin (I); brassinin (II); cyclobrassinin (III); spirobrassinin (IV) and glucosinolates were determined using high performance liquid chromatography procedures. Accumulation of phytoalexins I - III was evident within 8 h of irradiation, whilst formation of spirobrassinin was evident only after 24 h. Maximal levels of III and IV (> 100 μg g-1 freeze dried tissue) were greater than those of I and II (27 and 17 μg g-1, respectively). The individual glucosinolate levels were affected in a complex manner; whilst most glucoinolates decreased on storage, the levels of indole glucosinolates, glucobrassicin (XI) and 1-methoxyglucobrassicin (XIII), increased until 5 to 6 days after irradiation and thereafter declined. Whilst structural features of I - IV , XI and XIII suggest close biosynthetic relationships between these classes of biologically-active indoles, further studies are needed to establish this point unambiguously.

Sign in / Sign up

Export Citation Format

Share Document