Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-a-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of ¹⁴C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.     

Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia

Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.     

A simplified technique for producing an ischemic wound model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 °C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.     

Improvement in infected wound healing in type 1 diabetic rat by the synergistic effect of photobiomodulation therapy and conditioned medium

We investigated the effects of photobiomodulation therapy (PBMT) and conditioned medium (CM) of human bone marrow mesenchymal stem cells (hBM-MSC) individually and/or in combination on the stereological parameters and the expression of basic fibroblast growth factor (bFGF), hypoxia-inducible factor (HIF-1α), and stromal cell–derived factor-1α (SDF-1α) in a wound model infected with methicillin-resistant Staphylococcus aureus (MRSA) in diabetic rats. CM was provided by culturing hBM-MSCs. Type 1 diabetes mellitus (T1DM) was induced in 72 rats, divided into four groups, harboring 18 rats each: group 1 served as a control group, group 2 received PBMT, group 3 received CM, and group 4 received CM + PBMT. On days 4, 7, and 15, six animals from each group were euthanized and the skin samples were separated for stereology examination and gene expression analysis by real-time polymerase chain reaction. In the CM + PBMT, CM, and PBMT groups, significant decreases were induced in the number of neutrophils (1460 ± 93, 1854 ± 138, 1719 ± 248) and macrophages (539 ± 69, 804 ± 63, 912 ± 41), and significant increases in the number of fibroblasts (1073 ± 116, 836 ± 75, 912 ± 41) and angiogenesis (15 230 ± 516, 13 318 ± 1116, 14 041 ± 867), compared with those of the control group (2690 ± 371, 1139 ± 145, 566 ± 90, 12 585 ± 1219). Interestingly, the findings of the stereological examination in the CM + PBMT group were statistically more significant than those in the other groups. In the PBMT group, in most cases, the expression of bFGF, HIF-1α, and SDF-1α, on day 4 (27.7 ± 0.14, 28.8 ± 0.52, 27.5 ± 0.54) and day 7 (26.8 ± 1.4, 29.6 ± 1.4, 28.3 ± 1.2) were more significant than those in the control (day 4, 19.3 ± 0.42, 25.5 ± 0.08, 22.6 ± 0.04; day 7, 22.3 ± 0.22, 28.3 ± 0.59, 24.3 ± 0.19) and other treatment groups. The application of PBMT + CM induced anti-inflammatory and angiogenic activities, and hastened wound healing process in a T1 DM model of MRSA infected wound.     

Adult spinal opioid receptor μ1 expression after incision is altered by early life repetitive tactile and noxious procedures in rats

Clinical and experimental data suggests that noxious stimulation at critical stages of development results in long‐term changes on nociceptive processing in later life. Here, we use an established, well‐documented rat model of repetitive noxious procedures closely mimicking the clinical situation in the NICU. In order to understand molecular changes underlying the long‐term consequences of repetitive stimulation of the developing nociceptive system the present study aims to analyze the presence of the µ‐opioid‐receptor‐1 (OPRM1). Neonatal rats received either four needle pricks per day in the left hind‐paw from postnatal day 0–7 as a model of procedural pain in infancy. Control pups were handled in the same way but were instead tactile stimulated, or were left undisturbed. At the age of 8 weeks, all animals received an ipsilateral hind‐paw incision as a model for post‐operative pain, and mechanical sensitivity was tested at multiple time‐points. Before, and 1 or 5 days post‐incision, spinal cord tissue was collected for immunostaining of opioid receptor OPRM1. Semi‐quantitative immunocytochemical analysis of superficial laminae in lumbar spinal dorsal horn revealed that: (1) early life repetitive tactile or noxious procedures do not alter baseline levels of OPRM1 staining intensity and (2) early life repetitive tactile or noxious procedures lead to a decrease in OPRM1 staining intensity 5 days after incision in adulthood compared to undisturbed controls. We conclude that early life repetitive tactile or noxious procedures affect the intensity of OPRM1‐immunoreactivity in the lumbar superficial spinal cord dorsal horn after adulthood injury, without affecting baseline intensity. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 417–426, 2018     

Characterization of the high-affinity uptake of fructose-1,6-bisphosphate by cardiac myocytes

Previously, we reported that fructose-1,6-bisphosphate (FBP) was taken up by rat cardiac myocytes by two processes: a component that was saturable at micromolar levels and a nonsaturable component that dominated at millimolar levels. Here, we continued to characterize the saturable high-affinity component, with the aim of identifying the physiological substrate and role for this activity. ATP, ADP, and AMP inhibited the uptake of FBP with apparent affinities of 0.2-0.5 mM. Fumarate and succinate were very weak inhibitors. Several phosphorylated sugars (ribulose-1,5-phosphate, fructose-1-phosphate, ribose-5-phosphate, and inositol-2-phosphate) inhibited FBP uptake with apparent affinities of 40-500 μM. As in our previous study, no tested compound appeared to bind as well as FBP. The data suggest that the best ligands have two phosphoryl groups separated by at least 8 ?. The rates of FBP uptake were measured from 3° to 37°. The calculated activation energy was 15-50 kJ/mol, similar to other membrane transport processes. Uptake of FBP was tested in several types of cells other than cardiac myocytes, and compared to the uptake of 2-deoxyglucose and L: -glucose. While FBP uptake in excess of that of L: -glucose was observed in some cells, in no case was the uptake as high as in cardiac myocytes. The physiological substrate and role for the high-affinity FBP uptake activity remain unknown.     

Permeability of fructose-1,6-bisphosphate in liposomes and cardiac myocytes

Fructose-1,6-bisphosphate (FBP) helps preserve heart and other organs under ischemic conditions. Previous studies indicated that it can be taken up by various cell types. Here we extended observations from our group that FBP could penetrate artificial lipid bilayers and be taken up by cardiac myocytes, comparing the uptake of FBP to that of L-glucose. Using liposomes prepared by the freeze-thaw method, FBP entered about 200-fold slower than L-glucose. For liposomes of either soybean or egg lipids, 50 mM FBP enhanced the permeability of FBP itself, with little effect on general permeability (measured by uptake of L-glucose). In experiments with isolated cardiac myocytes at 21 degrees C, FBP uptake exceeded the uptake of L-glucose by several fold and appeared to equilibrate by 60 min. There was both a saturable component at micromolar levels and a nonsaturable component which dominated at millimolar levels. The saturable component was inhibited by Pi and by other phosphorylated sugars, though with lower affinity than FBP. Both saturable and nonsaturable uptakes were also observed at 3 degrees C. The results indicate that FBP enters myocytes not by simple penetration through the lipid bilayer, but via at least two distinct protein-dependent processes. The uptake could lead to intracellular effects important in hypothermic heart preservation.     

The uptake and metabolism of fructose‐1,6‐diphosphate in rat cardiomyocytes

Fructose1,6diphosphate (FDP) is a glycolytic intermediate which has been theorized to increase the metabolic activity of ischemic tissues. Here we examine the effects of externally applied FDP on cardiomyocyte uptake and metabolism. Adult rat cardiomyocytes were isolated and exposed to varying concentrations (0, 5, 25 and 50 mM) of FDP for either 1, 16 or 24 h of hypoxia (95% N₂/5% CO₂), each time period followed by a 1 h reoxygenation (95% air/5% CO₂). The uptake of FDP by rat cardiomyocytes was more concentrationdependent than timedependent. Furthermore, the uptake of FDP by the cardiomyocytes was similar in the hypoxia and normoxia treated cells. Alamar Blue, a redox indicator that is sensitive to metabolic activity, was used to monitor the effects of the FDP on cardiomyocyte metabolism. In the 1 h hypoxia or normoxia group, the 5, 10 and 25 mM FDP showed a significant increase in metabolism compared to the control cells. When the length of hypoxia was extended to 16 h, all doses of FDP were greater than control. And at the 24 h hypoxia or normoxia time period, only the 10, 25 and 50 mM FDP groups were greater than control. The results indicate a non-linear trend between the external concentration of FDP and the changes noted in metabolism. The findings from this study indicate that a narrow concentration range between 5–10 mM augments cardiomyocyte metabolism, but higher or lower doses may have little additional affect.