Physiological Buffer Saline Research Articles

Biocompatible fluorescent probe for detecting mitochondrial alkaline phosphatase activity in live cells

Alkaline phosphatase (ALP) is an enzyme that actively plays a significant role in the various metabolic processes by transferring a phosphate group to the protein, nucleic acid, etc. The elevated level of ALP in blood plasma is the hallmark of inflammation/cancer. The hyperactive mitochondria in cancer cells produce an excess of ATP to fulfill the high energy demand. Thus, we have developed a fluorescent probe Mito-Phos for ALP, which can detect phosphatase expression in mitochondria in live cells. The probe Mito-Phos has shown ~15-fold fluorescence intensity increments at 450 nm in the presence of 500 ng/mL of ALP. It takes about 60 min to consume the whole amount of ALP (500 ng/mL) in physiological buffer saline. It can selectively react with ALP even in the presence of other probable cellular reactive components. It is highly biocompatible and nontoxic to the live cells. It has shown ALP expression in a dose-dependent manner by providing concomitant fluorescence images in the blue-channel region. It has localized exclusively in the mitochondria in live cells. The probe Mito-Phos is highly biocompatible with the ability to assess ALP expression in mitochondria in live cells.

Immature bovine cartilage wear by fatigue failure and delamination

This study investigated wear damage of immature bovine articular cartilage using reciprocal sliding of tibial cartilage strips against glass or cartilage. Experiments were conducted in physiological buffered saline (PBS) or mature bovine synovial fluid (SF). A total of 63 samples were tested, of which 47 exhibited wear damage due to delamination of the cartilage surface initiated in the middle zone, with no evidence of abrasive wear. There was no difference between the friction coefficient of damaged and undamaged samples, showing that delamination wear occurs even when friction remains low under a migrating contact area configuration. No difference was observed in the onset of damage or in the friction coefficient between samples tested in PBS or SF. The onset of damage occurred earlier when testing cartilage against glass versus cartilage against cartilage, supporting the hypothesis that delamination occurs due to fatigue failure of the collagen in the middle zone, since stiffer glass produces higher strains and tensile stresses under comparable loads. The findings of this study are novel because they establish that delamination of the articular surface, starting in the middle zone, may represent a primary mechanism of failure. Based on preliminary data, it is reasonable to hypothesize that delamination wear via subsurface fatigue failure is similarly the primary mechanism of human cartilage wear under normal loading conditions, albeit requiring far more cycles of loading than in immature bovine cartilage.

Acute toxicity of the aqueous-methanolic Moringa oleifera (Lam) leaf extract on female Wistar albino rats

Background: Herbal preparations are widely assumed to be safe on oral administration and therefore the documentation of the toxic potential of some herbal concoctions used as medicine and nutrients is limited. Moringa oleifera ( MO ) is a plant that is gaining tremendous popularity in rural communities in Kenya as a means of offsetting nutritional and medicinal needs. However, very little is known about the safety of the plant on oral administration. Thus, the aim of the current study was to assess the biochemical and histological changes in the liver following the administration of an aqueous-methanolic (AQ-ME) MO leaf extract in female Wistar albino rats. Methods: Acute oral toxicity study on the AQ-ME MO leaf extract was conducted by the use of the limit test dose of the up and down procedure (OECD guideline number 425) with slight modifications. Briefly, ten (10) healthy, nulliparous, non-pregnant female Wistar strain albino rats aged 8-12 weeks and weighing 180±20 grams were used for the study. These animals were randomly selected into two groups; control and treatment group each having five (5) animals. They were then labelled to enable identification and control group animals were orally administered with physiological buffer saline once daily over a 48-hour period. The five (5) rats in the treatment group were dosed orally one at a time and once daily with a 2000 mg/kg dose of the AQ-ME MO leaf extract to determine the median lethal dose over a 48 hour period. Blood was then collected and used to prepare serum for biochemical analysis of aspartate amino transferase (AST), alanine amino transferase (ALT) and total bilirubin (TB) which are important biomarkers of liver dysfunction. Biochemical assays of these enzymes were performed using the method of the International Federation of Clinical Chemists (IFCC). Death was used as an endpoint, livers harvested and used to prepare transverse sections for histopathological examination. These sections were stained using the haematoxylin and eosin (H&E) method and observed for pathological changes using an optical microscope. Results: A 2000 mg/kg oral dose of AQ-ME MO leaf extract caused a significant (p 0.05) increase in the mean levels of total bilirubin in the treatment group relative to the control group. On the other hand, the extract caused a non-significant (p>0.05) decrease in the mean levels of ALT in the treatment group relative to the control. The post mortem analysis of the hepatic index (liver to body weight ratio) revealed that there was a non-significant increase (p>0.05) in the hepatic index of the treatment group relative to the control. However, the transverse liver sections of treatment group animals showed mild distortions in the architecture of liver cells. Conclusions: Based on these results, the LD 50 of the AQ-ME MO leaf extract was found to be >2000 mg/kg in female wistar albino rats.

Free DOX and chitosan-N-arginine conjugate stabilized indocyanine green nanoparticles for combined chemophotothermal therapy

Indocyanine green (ICG) is a FDA-approved near-infrared (NIR) cyanine dye used in medical diagnostics. However, the utility of ICG remains limited by its unstable optical property, and concentration-dependent aggregation and precipitation. A chitosan–arginine conjugate (CS-N-Arg) was developed to increase the stability of ICG in physiological buffer saline via formation of strong electrostatic interactions between ICG and CS-N-Arg. The CS-N-Arg/ICG complex prevented ICG from aggregation and precipitation, thus it could serve as a theranostic nanomaterial for image-guided photothermal cancer therapy. The CS-N-Arg/ICG NPs showed excellent photostability, clear fluorescent images, and rapid temperature rise under laser irradiation. Cell viability assay indicated that CS-N-Arg/ICG NPs could efficiently suppress the growth of doxorubicin (DOX) resistant breast cancer cell (MCF-7/ADR cells) under NIR photothermal treatments. In combination of DOX with CS-N-Arg/ICG NPs, a combined effect was observed in MCF-7/ADR breast cancer cells due to dual hyperthermia and chemical therapeutic effects. The present observations suggest that CS-N-Arg/ICG NPs can effectively deliver ICG molecules to MCF-7/ADR breast cancer cells and overcome DOX resistance in the cells by hyperthermia.

Humoral immune response of Salmonella typhimurium and Salmonella enteritidis sonicated antigens in rabbits

Salmonella typhimurium and salmonella enteritidis were isolated from infected goat andprepared an antigens of whole cell sonicated antigen of S.typhimurium(WCS.Ag.S.typhimurium ),whole cell sonicated antigen of S.enteritidis (WCS.Ag.S.entertidis) and combination of whole cell sonicated antigen (Salmonella typhimurium andSalmonella enteritidis) (CWS.Ag) . Their efficacy was evaluated by using tube agglutinationtest and enzyme linked immune sorbent assay (ELISA). Twenty rabbits were randomlydivided into four groups; the 1st group was immunized by WCS. Ag - Salmonella enteritidis,2nd group immunized by (WCS Ags .typhimurium), 3rd group immunized by CWCS.Agcompound and 4th left as control group which injected by physiological buffer saline (pH7.2). The antibody titer was increased in after the day 12, first, second and third months ofimmunization by agglutination test. IgG concentration was done by ELISA at the same time;which were recorded a higher significant differences (p˂ 0.01) at the first month in the groupimmunized by CWS Ag (449.65 ±38.6 1ng/ml IgG and 952± 20.85 antibodies titer )compared with other immunized groups ( WCS – Ag – S. enteritidis andWCS.Ag.S.typhimurium ). Also, the IgG concentration and antibodies titer are still higher inthe second and the third months in the immunized group by CWCS.Ag. 218.90± 6.69ng/ml,528± 68.58 and 89.55± 2.63ng/ml, 280± 49.98 respectively with significant differences (p˂0.01) compared with the immunized groups (WCS.Ag.S. entertidis and WCS. Ag.S.typhimurium) and also, they are significant (p˂ 0.01) when compared with the control groupResearch

Investigation of the frictional response of osteoarthritic human tibiofemoral joints and the potential beneficial tribological effect of healthy synovial fluid

This study tests the hypothesis that the natural progression of osteoarthritis (OA) in human joints leads to an increase in the friction coefficient. This hypothesis is based on the expectation that the wear observed in OA may be exacerbated by higher friction coefficients. A corollary hypothesis is that healthy synovial fluid (SF) may help mitigate the increase in the friction coefficient in diseased joints. The friction coefficient of human tibiofemoral joints with varying degrees of OA was measured in healthy bovine SF and physiological buffered saline (PBS). Two testing configurations were adopted, one that promotes sustained cartilage interstitial fluid pressurization to investigate the effectiveness of this mechanism with advancing OA, and another that allows interstitial fluid pressure to subside to investigate the effectiveness of boundary lubrication. Seven specimens were visually staged to be normal or mildly degenerated (stages< or =2 on a scale of 1 to 4) and nine others had progressive degeneration (stages>2 and< or =3). No statistical differences were found in the friction coefficient with increasing OA, whether in migrating or stationary contact area configurations; however, the friction coefficient was significantly lower in SF than PBS in both configurations. The friction coefficient of human tibiofemoral cartilage does not necessarily increase with naturally increasing OA, for visual stages ranging from 1 to 3. This outcome may be explained by the fact that interstitial fluid pressurization is not necessarily defeated by advancing degeneration. This study also demonstrates that healthy SF decreases the friction coefficient of OA joints relative to PBS.

Preparation and drug‐release behavior of minocycline‐loaded poly[hydroxyethyl methacrylate‐co‐poly(ethylene glycol)–methacrylate] films

AbstractIn this work, biocompatible hydrogel matrices for wound‐dressing materials and controlled drug‐release systems were prepared from poly[hydroxyethyl methacrylate‐co‐poly(ethylene glycol)–methacrylate] [p(HEMA‐co‐PEG–MA] films via UV‐initiated photopolymerization. The characterization of the hydrogels was conducted with swelling experiments, Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis (differential scanning calorimetry), and contact‐angle studies. The water absorbency of the hydrogel films significantly changed with the change of the medium pH from 4.0 to 7.4. The thermal stability of the copolymer was lowered by an increase in the ratio of poly(ethylene glycol) (PEG) to methacrylate (MA) in the film structure. Contact‐angle measurements on the surface of the p(HEMA‐co‐PEG–MA) films demonstrated that the copolymer gave rise to a significant hydrophilic surface in comparison with the homopolymer of 2‐hydroxyethyl methacrylate (HEMA). The blood protein adsorption was significantly reduced on the surface of the copolymer hydrogels in comparison with the control homopolymer of HEMA. Model antibiotic (i.e., minocycline) release experiments were performed in physiological buffer saline solutions with a continuous flow release system. The amount of minocycline release was shown to be dependent on the HEMA/PEG–MA ratio. The hydrogels have good antifouling properties and therefore are suitable candidates for wound dressing and other tissue engineering applications. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Prevention of Staphylococcus epidermidis biofilm formation using a low-temperature processed silver-doped phenyltriethoxysilane sol–gel coating

Sol–gel coatings which elute bioactive silver ions are presented as a potential solution to the problem of biofilm formation on indwelling surfaces. There is evidence that high-temperature processing of such materials can lead to diffusion of silver away from the coating surface, reducing the amount of available silver. In this study, we report the biofilm inhibition of a Staphylococcus epidermidis biofilm using a low-temperature processed silver-doped phenyltriethoxysilane sol–gel coating. The incorporation of a silver salt into a sol–gel matrix resulted in an initial high release of silver in de-ionised water and physiological buffered saline (PBS), followed by a lower sustained release for at least 6 days—as determined by graphite furnace-atomic absorption spectroscopy (GF-AAS). The release of silver ions from the sol–gel coating reduced the adhesion and prevented formation of a S. epidermidis biofilm over a 10-day period. The presence of surface silver before and after 24 h immersion in PBS was confirmed by X-ray photoelectron spectroscopy (XPS). These silver-doped coatings also exhibited significant antibacterial activity against planktonic S. epidermidis. A simple test to visualise the antibacterial effect of silver release coatings on neighbouring bacterial cultures is also reported.

Quantitative Determination of Enzyme Activity in Single Cells by Scanning Microelectrode Coupled with a Nitrocellulose Film-Covered Microreactor by Means of a Scanning Electrochemical Microscope

An electrochemical method for quantitative determination of enzyme activity in single cells was developed by scanning a microelectrode (ME) over a nitrocellulose film-covered microreactor with micropores by means of a scanning electrochemical microscope (SECM). Peroxidase (PO) in neutrophils was chosen as the model system. The microreactor consisted of a microwell with a solution and a nitrocellulose film with micropores. A single cell perforated by digitonin was injected into the microwell. After the perforated cell was lysed and allowed to dry, physiological buffer saline (PBS) containing hydroquinone (H2Q) and H2O2 as substrates of the enzyme-catalyzed reaction was added in the microwell. The microwell containing the extract of the lysed cell and the enzyme substrates was covered with Parafilm to prevent evaporation. The solution in the microwell was incubated for 20 min. In this case, the released PO from the cell converted H2Q into benzoquinone (BQ). Then, the Parafilm was replaced by a nitrocellulose film with micropores to fabricate the microreactor. The microreactor was placed in an electrochemical cell containing PBS, H2Q, and H2O2. After a 10-microm-radius Au ME was inserted into the electrochemical cell and approached down to the microreactor, the ME was scanned along the central line across the microreactor by means of a SECM. The scan curve with a peak was obtained by detecting BQ that diffused out from the microreactor through the micropores on the nitrocellulose film. PO activity could be quantified on the basis of the peak current on the scan curve using a calibration curve. This method had two obvious advantages: no electrode fouling and no oxygen interference.

High-Throughput Single-Cell Analysis for Enzyme Activity without Cytolysis

A novel high-throughput method without cytolysis for determination of enzyme activity inside single cells was developed by a combination of chemical cell perforation and an intracellular enzyme-catalyzed reaction. Peroxidase (PO) inside human neutrophils was chosen as the model system. Cells were perforated with digitonin to form micropores on the cell membrane. The perforated cells, with physiological buffer saline of pH 7.4 containing hydroquinone (H2Q) and H2O2, were continuously propelled by pressure through a capillary as the microsampler and microreactor. Small molecules H2Q and H2O2 could diffuse into the cell interior through the micropores on the cell membrane, and the large molecule PO remained in the cell interior. Intracellular PO converted H2Q into benzoquinone (BQ). BQ diffused out from the cell interior to the cell surface through the micropores and formed a BQ zone around the cell. The process proceeded in the capillary during cell movement. The BQ zones around every moving perforated cell were continuously delivered to the capillary outlet by hydraulic flow and detected. An average detection rate of >1 cell/min was obtained.

Measurement of Enzyme Activity in Single Cells by Voltammetry Using a Microcell with a Positionable Dual Electrode

The electrochemical single-cell analysis for enzyme activity was developed using microcells on a microcell array coupled with a positionable dual microelectrode. The microcell array with the nanoliter-scale microcells was constructed using simple chemical etching without photolithographic techniques. The positionable dual microelectrodes consisted of the nanometer-to-micrometer-radius Au disk working electrode and a approximately 80-microm-radius Ag/AgCl reference electrode. Peroxidase was chosen as the model enzyme. Factors that concern electrochemical single-cell analysis in microcells such as solution evaporation, interference of soluble oxygen, electrode size, solution volume, and electrode fouling were investigated and discussed. The 20 or 100 nL of detection volume was found to be suitable for peroxidase determination in single neutrophils or single acute promyelocytic leukemia cells without interference from intracellular macromolecules and electrode fouling, when the dual electrode with a 10-microm-radius Au disk working electrode was used. Cells were perforated with digitonin before transferring them into the microcells, to lyse cells easily. The perforated cells were transferred into the microcells by pushing a microscope slide on a drop of the cell suspension on the microcell array. After a single cell in the microcell was lysed using a freeze-thawing technique and allowed to dry, physiological buffer saline containing 2.0 x 10(-3) mol/L hydroquinone and 2.0 x 10(-3) mol/L H2O2 as the substrates of the enzyme-catalyzed reaction was added. The microcell array was positioned in a constant-humidity chamber to prevent evaporation. Then the dual electrode was inserted into the microcell by means of a scanning electrochemical microscope and the product benzoquinone of the enzyme-catalyzed reaction was voltammetrically detected. Peroxidase activity could be quantified using the steady-state current on the voltammogram after subtracting the blank and using the calibration curve.

Determination of glutathione in single human hepatocarcinoma cells by capillary electrophoresis with electrochemical detection

A method for determination of glutathione (GSH) in single human hepatocarcinoma (HH) cells was described by capillary zone electrophoresis with electrochemical detection at a gold/mercury amalgam micro-disk electrode. When HH cells were washed with the running buffer instead of physiological buffer saline, only one electrophoretic peak for GSH is depicted on the electropherograms of single HH cells. When electroosmotic injection of 0.01 mol/l NaOH for lysing the cell introduced into the capillary, the lysis time can be shorten to 5 s. The whole cell injection and no need of derivatization reaction lead more accurate and precise results. The average amount of GSH in an individual HH cell is 22.3±5.8 fmol (mean±standard deviation), which is consistent with that of its homogenate.

Neutrophil apoptosis during the resolution of bovine mammary gland injury

The role of neutrophil apoptosis in the resolution of bovine mammary gland injury induced by intramammary administration of physiological buffered saline (PBS) or lipopolysaccharide (LPS) was investigated. Twenty mammary glands of five non-pregnant heifers were used in the two studies and each animal received both stimuli. Samples of cell populations were collected by mammary gland lavages before and 24, 48, 72 and 96 hours after treatment and examined by light microscopy and staining for myeloperoxidase (MPO). A marked influx of neutrophils into the mammary gland was observed 24 hours after stimulation. At the same time, apoptotic neutrophils and MPO -positive macrophages (MAC) were identified in the samples. The numbers increased to reach maximum values at 48 hours after stimulation with PBS and at 72 hours after stimulation with LPS. The observed differences in the length of the resolution period indicate that neutrophil viability can be modulated by delaying the apoptotic process. Apoptosis of neutrophils and their subsequent phagocytosis byMAC can be regarded as a significant mechanism in the removal of neutrophils from the acutely injured mammary glands and, hence, in the resolution of bovine mastitis.

In vitro anthelmintic activity of root-tuber extract of Flemingia vestita, an indigenous plant in Shillong, India

The in vitro activity of root-tuber-peel extract of Flemingia vestita, an indigenous plant consumed by the natives in Northeast India, was tested against helminth parasites. Live parasites (nematode: Ascaris suum from pigs, A. lumbricoides from humans, Ascaridia galli and Heterakis gallinarum from domestic fowl; cestode: Raillietina echinobothrida from domestic fowl; trematode: Paramphistomum sp. from cattle) were collected in 0.9 % physiological buffered saline (PBS) and maintained at 37 +/- 1 degrees C. In vitro treatment of the parasites with the crude extract (50 mg/ml) in PBS revealed complete immobilization of the trematode and cestode in about 43 and 20 min, respectively. However, the cuticle-covered nematodes did not show any change in physical activity and remained viable even after a long period of exposure to the extract. Exposure of R. echinobothrida to genistein (0.5 mg/ml), an active principle isolated from the root-tuber peel, caused spontaneous loss of movement (paralysis) in 4.5 h, which was slower than the time required for praziquantel, the reference flukicide and cestodicide. The treated parasites showed structural alteration in their tegumental architecture. This study suggests the vermifugal activity of this plant extract against trematodes and cestodes.

Effects of heparin on vascularization of artificial skin grafts in rats

Artificial skin is a recent development in the clinical care of the severely burned patient. Its manufacture entails the covalent bonding of collagen and polysaccharide, followed by the coating of one surface with a thin layer of silicone rubber. Artificial skin was grafted onto rats and examined for neovascularization at 7 days. Vascular patency was shown by perfused yellow latex casts. Five percent of the patent vessels grew into the graft soaked in physiological buffered saline (PBS). When the graft was soaked in heparin, 1 mg/ml buffered saline solution, before grafting, 54% of the patent vessels in the grafted area had grown into the matrix. These experiments show that the local application of heparin promotes early ingrowth of blood vessels into the healing site. The vascularity of artificial skin can be modified by heparin, which promotes angiogenesis, and leads to earlier deposits of greater amounts of new connective tissue.