The use of Light Green and Orange II as quantitative protein stains, and their combination with the Feulgen method for the simultaneous determination of protein and DNA

The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and -Thionin(SO2) method for the simultaneous determination of DNA and protein. - With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. - In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. - When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. - When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.     

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy

The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1 M MgCl2, Cuprolinic Blue specifically stains single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.     

Lack of selectivity between anesthetic stereoisomers for an inhibitory site which is located on a membrane protein

Lack of selectivity towards anesthetic stereoisomers is one of the few criteria available for the identification of an anesthetic target site. Until now this criterion has not been tested for anesthetics that directly interact with sensitive membrane proteins which are considered the targets of anesthetic action. In this communication we show that stereoisomers of 2-butanol and 2,4-pentanediol did not differ in their inhibitory potency for a site located on the Na+/K+/Cl- co-transporter protein. This suggests that an inhibition site on a membrane protein can fulfill the criterion of lack of stereoisomer selectivity.     

Crucial markers showing the risk of coronary artery disease in obesity: ADMA and neopterin

BackgroundObesity is responsible for high morbidity and mortality, both in developed and developing countries. It is associated with many chronic and metabolic diseases. Asymmetric dimethylarginine (ADMA) has been demonstrated to be a biomarker of endothelial dysfunction in humans and increased ADMA associated with cardiovascular disease (CVD) risk has been reported in many states. Neopterin (NP) produced by monocytes/macrophages in response to stimulation by interferon-gamma (IFN-γ) is emphasized in recent findings. The current study aims to investigate ADMA and NP levels which may assume a role in guiding the early diagnosis of coronary artery disease in obesity.MethodsThis is an original research study in which ADMA and NP levels of 50 patients (25 male/25 female) diagnosed with obesity were compared with those of 30 healthy individuals (15 male/15 female) as control. The high-performance liquid chromatography (HPLC) method was used while determining parameters.ResultsADMA and NP levels in obese individuals were found to be significantly higher than in those enrolled in the control. ADMA values were found to be higher in obese subjects (0.71±0.24 μmol/L) as compared with levels found in healthy subjects (0.58±0.16 μmol/L) (p<0.05). A significant increase of serum neopterin levels was found in obese subjects (8.8±3.5 μmol/L) as compared with controls (4.9±1.69 μmol/L) (p<0.05). Also, there was a strong positive correlation between NP and ADMA values in obese individuals (r=0.954).ConclusionsOur study revealed that obese subjects have higher ADMA and neopterin levels. These results demonstrated that both ADMA and NP levels may be potential risk factors for coronary heart disease in obesity.     

Detection of metabolic changes in hepatocytes by quantitative cytochemistry

Studies by means of quantitative histochemistry and cytochemistry have greatly contributed to the knowledge of metabolic changes in liver parenchymal cells. In the present paper recent work along this line is reviewed with emphasis on three topics, polyploidy as a source of metabolic heterogeneity, proteolysis in the regulation of hepatocyte cell mass and ischemic injury of hepatocytes. In all three fields, accuracy and precision of information obtained by quantitative histochemical means has been greatly enhanced by a thorough knowledge of the mechanisms of histochemical reactions obtained by fundamental work on matrix chemistry, and well-considered application of optical measuring tools and conditions of measurement. These are the principles put forward by van Duijn since the pioneer period of histochemistry and to whom this review is dedicated.     

From the freezer to the clinic: Antifreeze proteins in the preservation of cells,tissues, and organs

Understanding the mechanisms by which natural anti‐freeze proteins protect cells and tissues from cold could help to improve the availability of donor organs for transplantation.

The first successful organ transplant in humans was performed in 1954 by Joseph Murray, who used a patient’s twin as a kidney donor. Murrays’ breakthrough paved the way for organ transplantation and the number of transplanted organs has grown ever since. For example, in 2017, a total of 139.024 solid organs—mostly kidney, liver, heart, lung, pancreas, and small bowel—were transplanted (Fig 1A). But this number only reflects 10% of the worldwide need; many patients still die of end‐stage organ failure while on a waiting list. The limited number of donor organs contributes only partially to this shortage. Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime. In fact, the percentage of donor organs that are unused is estimated to range from around 25% for kidneys and livers up to 70–80% for hearts and lungs (Giwa et al, 2017); Fig 1B).Open in a separate windowFigure 1Organ transplantation and preservability statusStatistics show a positive correlation between the duration of ex vivo preservation and the number of organ transplants. Number of solid organs transplanted in 2017 (A). Percentage of organs failed to be transplanted (B). Duration of solid organ ex vivo preservation in static cold storage (C). Sources: Data from the Global Observatory on Donation and Transplantation and (Parsons et al, 2014), (Guibert et al, 2011) and (Editorial: Buying time for transplants (2017))

Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime.

To address the shortage of donor organs and decrease the number of organs that go to waste, biobanks could efficiently store viable tissues and organs until transplantation. Yet, the current standard for ex vivo preservation of donor organs is static cold storage (4–8°C) which, depending on the organ, ensures viable conservation for only some hours; hearts are typically viable for a maximum of only 4 h (Fig 1C). In addition, this approach leads to hypothermic damage and to ischemia/reperfusion injury.Hence, there is an urgent need for strategies that prolong the viable preservation of donor organs. Two main strategies have emerged for cryopreservation and subzero storage, both of which cool tissues below the freezing point. While subzero storage just below 0°C may suffice for short‐term preservation, cryopreservation at −80°C or even lower temperatures is required for long‐term storage in biobanks. A proof‐of‐principle study already demonstrated that subzero preservation extended the preservation of rat hearts up to 24 h after collection (Amir et al, 2004); cryopreservation of whole hearts is currently not possible. The main reason is that lowering the temperature below the freezing point of water leads to ice formation, which causes cell damage and destroys tissues. One of the main challenges in biomedical research for organ transplantation is therefore finding non‐toxic and biocompatible antifreeze compounds that enable subzero storage and cryopreservation without causing tissue damage. An additional benefit is a larger time window to perform evaluation in terms of organ size and human leukocyte antigens matching and preparing the recipient patient to increase the chance of a successful transplantation.