Membrane surface properties of sheep erythrocytes,an immunological reagent,after different treatments as reflected by partition in two-polymer aqueous phases

Sheep erythrocytes (E) which, with or without certain treatments, are currently used as “immunological reagents” to detect cells with specific receptors (by rosette-formation) have been partitioned in two-polymer aqueousphase systems selected so as to reflect charge-associatedor lipid-related membrane surface properties. We have found that the partitioning behavior of E is not affected in these phases by reacting the cells with anti-E antibody (either IgG or IgM), forming EA. The additional binding of complement to the cell-antibody complex, forming EAC, results, however, in a marked decrease in the partition coefficient,K. Apparently both the charge-associated and hydrophobic properties reflected by partitioning remain accessible to the phase polymers when the cells are coated with antibody, but are not with the addition of complement. It is interesting that EA can still rosette with T-lymphocytes (14), a property of E, while the additional coating with complement results in EAC which does not appreciably do so (26). Neuraminidase or trypsin treatments of E, which yield Es having quite different rosetting properties with T-lymphocytes (14), cause increasedKs and unchangedKs, respectively, in phases reflecting lipid-related surface properties. Either treatment causes reducedKs of E in charged-phase systems. Neuraminidase treatment also results in a reduced electrophoretic mobility of E, while trypsin treatment is not detectable by cell electrophoresis (25). We are currently studying the possible usefulness of employing cell electrophoresis and cell partitioning in charged-phase systems jointly to obtain information on events occurring at the shear plane versus those occurring deeper in the membrane.     

Diazocyclopentadiene (DACP), a light sensitive reagent for the ethylene receptor in plants

Diazocyclopentadiene (DACP) has been shown to be an effective reagent for the ethylene receptor. Treatment of mung bean sprouts or tobacco leaves with DACP in the light or in the dark inactivates much of the ethylene binding. In the light, inactivation seems to be permanent, while in the dark, the site becomes active again after the DACP diffuses away. The compound is 10 times more effective in the light than in the dark. DACP inhibits banana ripening indicating the physiological receptor is involved. It also overcomes the inhibitory effect of ethylene on mung bean seedling growth (Km = 0.09 µl/1 E) at low ethylene levels. At high ethylene levels, an apparent high ethylene level site becomes apparent (Km = 50 µl/1 E) and growth is inhibited.     

The Recognition of Lipolytic Activity with the Microscope

In view of its probable wide applicability, it seems desirable to publish a note on a simple technic for the recognition with the microscope of the action of lipase. In brief, the method is to make an emulsion of neutral fat previously stained with a red Sudan stain, subject some of the emulsion to the action of the supposed lipase for an appropriate time and then examine with the microscope a recovered drop of the emulsion in a solution of Nile blue sulfate. It has long been known that Nile blue sulfate stains liquid neutral fats a reddish color and fatty acids blue.     

Characterisation and computational analysis of a novel lipase nanobio-based reagent for visualising latent fingerprints on water-immersed glass slides

Considering the significant evidential values of fingerprints in underwater criminal investigations and the need to visualise them using a user- and environmentally-friendly reagent, development of a novel, rapid and relatively greener nanobio-based reagent (NBR) is deemed beneficial. Lipase from the commercial Candida rugosa immobilised onto acid-functionalised multi-walled carbon nanotubes (NBR) was used as the safer and cheap lipid-sensing reagent to visualise groomed whole/split fingerprints on non-porous objects immersed in stagnant tap water for up to 30 days under a laboratory-controlled setting. Attenuated Total Reflectance – Fourier Transform Spectrometry, Field Emission Scanning Electron Microscopy and bioinformatics (molecular docking and molecular dynamics simulations) were employed to characterise and confirm the attachment of NBR onto the lipid constituents of wet fingerprints. Chromatographic results further confirmed the presence of n-hexadecanoic and octadecanoic acids on fingerprints up to 30 days of immersion. Thus, NBR may potentially be useful as the future state-of-the-art fingerprint visualisation technology.     

Synthesis and anti-proliferative activity of a novel 1,2,3-triazole tethered chalcone acetamide derivatives

A new series of 1,2,3-triazole tethered chalcone acetamide derivatives (7a-c & 8a-r) have been synthesized in excellent yields and their structures were determined by analytical and spectral (FT-IR, ¹H NMR, ¹³C NMR & HRMS) studies. The newly synthesized derivatives were evaluated for their cytotoxic activity against four human cancer cell lines, such as HeLa (Human cervical cancer), A549 (Human alveolar adenocarcinoma), MCF-7 (Human breast adenocarcinoma) and SKNSH (Human brain cancer). Among them, compound 7c exhibited good anti-proliferation activity with HeLa (IC₅₀ 7.41 + 0.8 μM), SKNSH (IC₅₀ 8.68 + 1.1 μM), MCF-7 (IC₅₀ 9.76 + 1.3 μM) and MDA-MB-231, while compounds 7a and 7b showed promising anti-proliferation against above four human cancer cell lines with IC₅₀ 7.95–11.62 μM, respectively, compared with the standard drug Doxorubicin. We explored the probable key active site and binding mode interactions in HDAC8 (PDB ID:3SFH) and EHMT2 (PDB ID:3K5K) proteins. The docking results are complementary to the experimental observations.     

C/N驱动优势细菌菌群变化影响堆肥碳氮损失和腐殖质合成

为了探明C/N如何驱动堆肥过程中优势细菌菌群的变化而影响碳氮损失和腐殖质合成,设置3个C/N处理(20∶1、25∶1和30∶1),以羊粪和玉米秸秆为原料进行堆肥试验。结果表明: 与20∶1处理相比,30∶1和25∶1处理堆肥的碳、氮损失分别降低了33.5%、18.9%和23.6%、10.8%。优势细菌菌群、碳氮损失及有机碳组分的冗余分析表明,高C/N提高了堆肥中固氮细菌的种类和丰度,降低了反硝化细菌的种类和丰度,减少了堆肥过程中的碳氮损失;高C/N促进了木质纤维素类降解菌的生长繁殖,促进了富里酸和胡敏素降解而合成更多胡敏酸,提高了堆肥腐殖化程度。可见,C/N可通过影响堆肥中关键优势细菌菌群而影响堆肥过程和堆肥质量,调节堆肥原料C/N可以调控堆肥中碳氮损失和腐殖质的合成,从而提高堆肥质量并减少堆肥的二次环境污染。     

Standardization in Biological Staining. The Influence of Dye Manufacturing

The purpose of biological staining is to obtain specimens of biological material that can be assessed in the microscope. These specimens are influenced by all processes from removal from the intact organism to mounting on the microscopic slide. To achieve comparable results with various techniques for biological staining, standardization of all procedures and reagents is mandatory. In this paper, I focus particularly on dyes and consider the possibilities for obtaining standardized dyes. In general practice, most biological staining takes place with available commercial dyes. These dyes may or may not have been subjected to quality assessment either internally by the producer or vendor or externally by independent investigators or organizations such as the Biological Stain Commission. Concerted attempts at standardization in Europe are discussed. The latest results of this work, the European standard EN 12376, is presented. This standard is concerned with information supplied by the manufacturer with in vitro diagnostic reagents for biological staining. The standard has been prepared by a Working Group on Staining in Biology under Technical Committee 140, In Vitro Medical Devices, of the European committee for standardization, CEN.     

PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.     

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.     

Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules

Legumes form a symbiotic interaction with Rhizobiaceae bacteria, which differentiate into nitrogen‐fixing bacteroids within nodules. Here, we investigated in vivo the pH of the peribacteroid space (PBS) surrounding the bacteroid and pH variation throughout symbiosis. In vivo confocal microscopy investigations, using acidotropic probes, demonstrated the acidic state of the PBS. In planta analysis of nodule senescence induced by distinct biological processes drastically increased PBS pH in the N₂‐fixing zone (zone III). Therefore, the PBS acidification observed in mature bacteroids can be considered as a marker of bacteroid N₂ fixation. Using a pH‐sensitive ratiometric probe, PBS pH was measured in vivo during the whole symbiotic process. We showed a progressive acidification of the PBS from the bacteroid release up to the onset of N₂ fixation. Genetic and pharmacological approaches were conducted and led to disruption of the PBS acidification. Altogether, our findings shed light on the role of PBS pH of mature bacteroids in nodule functioning, providing new tools to monitor in vivo bacteroid physiology.