Intralysosomal accumulation of polyanions. I. Fusion of pinocytic and phagocytic vacuoles with secondary lysosomes.

The long-term exposure of macrophages to low concentrations of a number of polyanions leads to their accumulation in high concentration within secondary lysosomes. This was associated with enlargement of the lysosomes, the presence of membranous whorls, and intense toluidine blue staining of the organelles at pH 1.0. After the ingestion of a particulate load by these cells, newly formed phagocytic vacuoles failed to fuse with polyanion-laden lysosomes. The lack of fusion was evident in both fluorescence and electron micrographic studies which followed the transfer of acridine orange or Thorotrast from 2 degrees lysosomes to phagosomes. Agents that inhibited phagosome-lysosome (P-L) fusion included molecules containing high densities of sulfate, sulfonate, or carboxylate residues. Dextran sulfate (DS) in microgram/ml quantities was an excellent inhibitor, whereas nonsulfated dextran (D) was without effect at 1,000-fold higher concentrations. In contrast to their effects on P-L fusion, polyanions failed to influence the fusion of pinocytic vesicles with 2 degrees lysosomes. The uptake, intravacuolar distribution, and intralysosomal digestion of fluid-phase pinocytic markers were unaltered in lysosomes containing either D or DS. Furthermore, subcellular fractionation studies showed that the fluid-phase pinocytic marker HRP was efficiently transferred from pinosomes to large, dense 2 degrees lysosomes containing DS.

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Renal hemodynamic and excretory responses to intra-arterial infusion of peroxynitrite in anesthetized rats

AJP Renal Physiology ◽

10.1152/ajprenal.90487.2008 ◽

2009 ◽

Vol 296 (1) ◽

pp. F170-F176 ◽

Cited By ~ 10

Author(s):

Luis C. Matavelli ◽

Philip J. Kadowitz ◽

L. Gabriel Navar ◽

Dewan S. A. Majid

Keyword(s):

Low Dose ◽

No Synthase ◽

High Concentration ◽

Arterial Infusion ◽

Anesthetized Rats ◽

Low Concentrations ◽

Nos Inhibitor ◽

Renal Responses ◽

Higher Doses

Peroxynitrite (ONOO−) is formed endogenously by the reaction of nitric oxide (NO) and superoxide (O2−). To examine the hypothesis that OONO− cause renal vasodilation at low concentrations but cause vasoconstriction at higher concentrations, we examined renal responses to intra-arterial infusion of incremental doses of OONO− (10, 20, and 40 μg·kg−1·min−1; 45 min each) in anesthetized rats. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearance. In control rats ( n = 6), low dose (10 μg·kg−1·min−1) of OONO− increased RBF by 10 ± 3% and GFR by 15 ± 5%. The higher doses (20 and 40 μg·kg−1·min−1) mostly reversed these responses which were −7 ± 4 and −27 ± 7% ( P < 0.05) in RBF and −0.1 ± 4.8 and −14 ± 12% in GFR, respectively. There were no appreciable changes in urine flow (V) and sodium excretion (UNaV) during OONO− infusion. However, in rats pretreated with NO synthase (NOS) inhibitor, l-NAME (50 μg·kg−1·min−1; n = 5), these doses of ONOO− significantly reduced RBF (−26 ± 7, −27 ± 6, and −44 ± 3%) and GFR (−21 ± 6, −25 ± 8, and −32 ± 12%) with variable increases in V or UNaV. Long-term infusion of OONO− (10 μg·kg−1·min−1 for 75 min) in another set of control rats ( n = 5) also showed similar vasodilator and hyperfiltration responses. These data indicate that ONOO− acts as an oxidant at high concentration but provides renoprotective function at low concentration that depends on intact NOS activity.

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The membrane proteins of the vacuolar system. II. Bidirectional flow between secondary lysosomes and plasma membrane.

The Journal of Cell Biology ◽

10.1083/jcb.86.1.304 ◽

1980 ◽

Vol 86 (1) ◽

pp. 304-314 ◽

Cited By ~ 87

Author(s):

W A Muller ◽

R M Steinman ◽

Z A Cohn

Keyword(s):

Plasma Membrane ◽

Culture Medium ◽

Fluid Phase ◽

Membrane Flow ◽

Em Autoradiography ◽

Secondary Lysosomes ◽

Bidirectional Flow ◽

Vacuolar System ◽

Representative Samples ◽

Pinocytic Vesicles

Lactoperoxidase covalently coupled to latex spheres (LPO-latex) has been used to selectively iodinate the phagolysome (PL) membrane within living macrophages, as discussed in the accompanying article. This procedure labeled approximately 24 polypeptides in the PL membrane; these were similar to those iodinatable on the external surface of the plasma membrane (PM). We now report on the translocation and fate of these proteins when the cells are returned to culture. TCA-precipitable radioactivity was lost from cells with biphasic kinetics. 20-50% of the cell-associated radiolabel was rapidly digested (t 1/2 approximately equal to 1 h) and recovered in the culture medium as monoiodotyrosine. 50-80% of the label was lost slowly from cells ( 1/2 approximately equal to 24-30 h). Quantitative analysis of gel autoradiograms showed that all radiolabeled proteins were lost at the same rate in both the rapid and slow phases of digestion. Within 15-30 min aftr labeling of the PL membrane, EM autoradiography revealed that the majority of the cell-associated grains, which at time 0 were associated with PL, were now randomly dispersed over the plasmalemma. At this time, analysis of PM captured by a second phagocytic load revealed the presence of all labeled species originally present in the PL membrane. This demonstrated the rapid, synchronous centrifugal flow of PL polypeptides to the cell surface. Evidence was also obtained for the continuous influx of representative samples of the PM into the PL compartment by way of pinocytic vesicles. This was based on the constant flow of fluid phase markers into latex-containing PL and on the internalization of all iodinatable PM polypeptides into this locus. These observations provide evidence for the continuous, bidirectional flow of membrane polypeptides between the PM and the secondary lysosome and represent an example of a membrane flow and recycling mechanism.

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Intake of different hays with presence of Colchicum autumnale by rabbits

World Rabbit Science ◽

10.4995/wrs.2017.6486 ◽

2017 ◽

Vol 25 (1) ◽

pp. 51

Author(s):

T. Eve ◽

L. Nief ◽

S. Gonçalves ◽

C. Fontfreyde ◽

S. Jurjanz

Keyword(s):

Animal Health ◽

High Concentration ◽

Colchicum Autumnale ◽

Aerial Parts ◽

Commercial Feed ◽

Low Concentrations ◽

Real Value ◽

Rabbit Meat ◽

Toxic Plant

The ingestion of 3 hay types by growing rabbits and the consequences on animal performance were studied in a first part. Two kinds of hay from high nature value grasslands [CFP (Colchico-Festucetum pratensis) and SBR (Senecioni-Brometum racemosi)] differentiated by their botanical composition were compared to a classical agricultural hay (control). The 3 hays, offered ad libitum, were ingested at 30.6, 34.8 and 36.1 g/kg body weight (BW) respectively for SBR, CFP and the control, where the consumption of SBR hay was significantly lower. (P<0.01). The type of hay distributed did not affect the consumption of commercial feed or growth (44 g/kg BW and 36 g/d, respectively). The effect of meadow saffron concentration (Colchicum autumnale) in CFP hay was tested at 3 doses (absence, 2.75 and 15% of the distributed hay mass) with respect to their effect on ingestion and animal health. Again, no effect of the presence of meadow saffron (MeS) in the hay on these parameters was observed, even at the highest dose. However, during this second part, rabbits preferred to consume meadow saffron instead of the rest of the distributed hay (hardly any MeS was refused by rabbits at the representative dose, whereas 12% of the rest of the hay was refused, and 3.4 and 20% respectively at the maximal dose). The intake of the toxic alkaloid colchicine corresponded to 30 to 44% of the median lethal doses (LD50) at the representative and maximal doses respectively. The real exposure of the rabbits was probably lower, as the absorption of ingested colchicine is reported to be around 50% of the LD50 values which are established after an intravenous injection. Nevertheless, the absence of any effect should be interpreted with caution. The colchicine content of MeS varied greatly depending on the time of harvest and the parts of the plant taken into consideration. A late harvest (mid-June or later) would ensure (very) low concentrations of colchicine in the aerial parts of MeS. The presence of cloves on this plant should be completely avoided, due to the very high concentration of colchicine in the seeds. Moreover, long term exposure of the animals to this toxic plant may increase the risk of adverse effects. The study shows that hay distribution can aid digestion without altering the animal’s performance. Moreover, the valorisation of hay from high nature value grasslands would not only add real value to the commercialisation of rabbit meat by commercial breeders, but also to pet owners on the condition that hay quality would be guaranteed.

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Anatomical and biochemical studies of Spartium junceum infected by Xylella fastidiosa subsp. multiplex ST 87

PROTOPLASMA ◽

10.1007/s00709-021-01640-2 ◽

2021 ◽

Author(s):

S. Falsini ◽

C. Tani ◽

G. Sambuco ◽

A. Papini ◽

P. Faraoni ◽

...

Keyword(s):

Xylella Fastidiosa ◽

Blue Staining ◽

Bacterial Cells ◽

High Concentration ◽

Toluidine Blue Staining ◽

Outbreak Area ◽

Phenolic Components ◽

The Matrix ◽

First Time ◽

Spartium Junceum

AbstractSpartium junceum L. is a typical species of Mediterranean shrubland areas, also grown in gardens and parks as an ornamental. In recent years in Europe, S. junceum has been recurrently found to be infected by different subspecies and genotypes of the quarantine regulated bacterium Xylella fastidiosa (Xf). This work presents for the first time the anatomy of S. junceum plants that we found, by means of genetic and immunochemistry analysis, to be naturally infected by Xf subsp. multiplex ST87 (XfmST87) in Monte Argentario (Grosseto, Tuscany, Italy), a new outbreak area within the EU. Our anatomical observations showed that bacteria colonized exclusively the xylem conductive elements and moved horizontally to adjacent vessels through pits. Interestingly, a pink/violet matrix was observed with Toluidine blue staining in infected conduits indicating a high content of acidic polysaccharides. In particular, when this pink-staining matrix was observed, bacterial cells were either absent or degenerated, suggesting that the matrix was produced by the host plant as a defense response against bacterial spread. In addition, a blue-staining phenolic material was found in the vessels and, at high concentration, in the pits and inter-vessels. SEM micrographs confirmed that polysaccharide and phenolic components showed different structures, which appear to be related to two different morphologies: fibrillary and granular, respectively. Moreover, our LM observations revealed bacterial infection in xylem conductive elements of green shoots and leaves only, and not in those of other plant organs such as roots and flowers.

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Increase associated risk of gynaecological cancer due to long-term exposure to high concentration of atmospheric SO2 industrial pollutant

Indoor and Built Environment ◽

10.1177/1420326x211003655 ◽

2021 ◽

pp. 1420326X2110036

Author(s):

Qian Xu ◽

Chan Lu ◽

Rachael Gakii Murithi ◽

Lanqin Cao

Keyword(s):

Time Windows ◽

Air Pollutant ◽

Gynaecological Cancer ◽

High Concentration ◽

The Past ◽

Short And Long Term ◽

Using Data ◽

Air Quality Monitoring Stations ◽

Control Study

A cohort case–control study was conducted in XiangYa Hospital, Changsha, China, which involved 305 patients and 399 healthy women, from June 2010 to December 2018, to evaluate the association between Chinese women’s short- and long-term exposure to industrial air pollutant, SO2 and gynaecological cancer (GC). We obtained personal and family information from the XiangYa Hospital electronic computer medical records. Using data obtained from the air quality monitoring stations in Changsha, we estimated each woman’s exposure to the industrial air pollutant, sulphur dioxide (SO2), for different time windows, including the past 1, 5, 10 and 15 years before diagnosis of the disease. A multiple logistic regression model was used to assess the association between GC and SO2 exposure. GC was significantly associated with long-term SO2 exposure, with adjusted odds ratio (95% confidence interval) = 1.56 (1.10–2.21) and 1.81 (1.07–3.06) for a per interquartile range increase in the past 10 and 15 years, respectively. Sensitivity analysis showed that different groups reacted in different ways to long-term SO2 exposure. We concluded that long-term exposure to high concentration of industrial pollutant, SO2 is associated with the development of GC. This finding has implications for the prevention and reduction of GC.

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Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes

Biology of Reproduction ◽

10.1093/biolre/ioab059 ◽

2021 ◽

Author(s):

Yasuyoshi Fukuda ◽

Misako Higashiya ◽

Takahiro Obata ◽

Keita Basaki ◽

Megumi Yano ◽

...

Keyword(s):

Cooling Rate ◽

Small Volume ◽

Sucrose Solution ◽

High Concentration ◽

Rat Embryos ◽

Low Concentrations ◽

Intracellular Ice Formation ◽

Intracellular Ice ◽

Warming Rate ◽

Very High

Abstract To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

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Self-buffering capacity of a human sulfatase for central nervous system delivery

Scientific Reports ◽

10.1038/s41598-021-86178-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yi Wen ◽

Nazila Salamat-Miller ◽

Keethkumar Jain ◽

Katherine Taylor

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Buffering Capacity ◽

The Self ◽

High Concentration ◽

Formulation Design ◽

The Central Nervous System ◽

Therapeutic Enzymes ◽

Intrathecal Space

AbstractDirect delivery of therapeutic enzymes to the Central Nervous System requires stringent formulation design. Not only should the formulation design consider the delicate balance of existing ions, proteins, and osmolality in the cerebrospinal fluid, it must also provide long term efficacy and stability for the enzyme. One fundamental approach to this predicament is designing formulations with no buffering species. In this study, we report a high concentration, saline-based formulation for a human sulfatase for its delivery into the intrathecal space. A high concentration formulation (≤ 40 mg/mL) was developed through a series of systematic studies that demonstrated the feasibility of a self-buffered formulation for this molecule. The self-buffering capacity phenomenon was found to be a product of both the protein itself and potentially the residual phosphates associated with the protein. To date, the self-buffered formulation for this molecule has been stable for up to 4 years when stored at 5 ± 3 °C, with no changes either in the pH values or other quality attributes of the molecule. The high concentration self-buffered protein formulation was also observed to be stable when exposed to multiple freeze–thaw cycles and was robust during in-use and agitation studies.

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Pediatric Antiphospholipid Syndrome: from Pathogenesis to Clinical Management

Current Rheumatology Reports ◽

10.1007/s11926-020-00976-7 ◽

2021 ◽

Vol 23 (2) ◽

Author(s):

Silvia Rosina ◽

Cecilia Beatrice Chighizola ◽

Angelo Ravelli ◽

Rolando Cimaz

Keyword(s):

Antiphospholipid Syndrome ◽

Fluid Phase ◽

Multiple Organ ◽

Clinical Settings ◽

Glycoprotein I ◽

Long Term Follow Up ◽

Pediatric Populations ◽

Cellular Components

Abstract Purpose of Review Elucidating the pathogenic mechanisms mediated by antiphospholipid antibodies (aPL) might exert important clinical implications in pediatric antiphospholipid syndrome (APS). Recent Findings aPL are traditionally regarded as the main pathogenic players in APS, inducing thrombosis via the interaction with fluid-phase and cellular components of coagulation. Recent APS research has focused on the role of β2 glycoprotein I, which bridges innate immunity and coagulation. In pediatric populations, aPL should be screened in appropriate clinical settings, such as thrombosis, multiple-organ dysfunction, or concomitant systemic autoimmune diseases. Children positive for aPL tests often present non-thrombotic non-criteria manifestations or asymptomatic aPL positivity. In utero aPL exposure has been suggested to result in developmental disabilities, warranting long-term follow-up. Summary The knowledge of the multifaceted nature of pediatric APS should be implemented to reduce the risk of underdiagnosing/undertreating this condition. Hopefully, recent pathogenic insights will open new windows of opportunity in the management of pediatric APS.

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Ultrafast Toluidine Blue Staining for Rapid On-Site Evaluation of Cytological Smears

Acta Cytologica ◽

10.1159/000505254 ◽

2020 ◽

Vol 64 (4) ◽

pp. 375-377

Author(s):

Ekkehard Hewer ◽

Anja M. Schmitt

Keyword(s):

Organic Solvents ◽

Toluidine Blue ◽

Critical Factor ◽

Blue Staining ◽

Nuclear Morphology ◽

Hazardous Chemicals ◽

Toluidine Blue Staining ◽

Site Evaluation ◽

The Rose

Rapid on-site evaluation (ROSE) is one of cytopathology’s “unique selling propositions.” The quality, speed, and ease of handling of the staining used is a critical factor for the efficacy of the ROSE procedure. Here, we describe a modification of rapid toluidine blue staining that can be performed within 25 s, provides excellent nuclear morphology, and is compatible with subsequent Papanicolaou staining of the slides. Furthermore, exposure to hazardous chemicals is minimized, as no organic solvents other than the alcohol-based fixative and glycerin for temporary mounting and coverslipping are required. We have used this protocol successfully in our ROSE practice and have not observed any discrepancies between toluidine blue- and permanent Papanicolaou-stained slides.

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Indoor radon concentration and a diffusion model in dwellings situated in a subalkaline granitoid area, Southern Brazil

Environmental Earth Sciences ◽

10.1007/s12665-021-09849-3 ◽

2021 ◽

Vol 80 (17) ◽

Author(s):

G. Romero-Mujalli ◽

A. Roisenberg ◽

A. Cordova-Gonzalez ◽

P. H. P. Stefano

Keyword(s):

Diffusion Model ◽

Indoor Radon ◽

Southern Brazil ◽

High Concentration ◽

Indoor Radon Concentration ◽

One Dimensional ◽

Environmental Limits ◽

Radon Measurements ◽

Cr 39 Detectors

AbstractRadon (Rn), a radioactive element, has especial interest in medical geology because long-term exposure to high concentration is related to lung cancer. In this study, outdoor and indoor radon measurements were conducted in dwellings of the Piquiri Syenite Massif, located in southern Brazil, given the relative high Rn content in soils of this region. Measurements were done using CR-39 detectors and placing them inside and outside dwellings. Moreover, a one-dimensional diffusion model was performed in order to quantify the natural transport of Rn to the air in confined and aerated environments. Results indicate that the region presents relatively low air Rn concentrations, within the environmental limits; however, the health risk might increase in confined and ill-ventilated environments because of transfer from soil and exhalation from ornamental rock-material often found inside dwellings. The main north facies of the syenite, where most of the rock extractions are located, was found to have the highest air Rn concentration because of the higher soil Rn concentration, compared to other facies of the syenite.

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