Injection Of Buffer Research Articles

Decelerated neurodegeneration after intravitreal injection of \u03b1-synuclein antibodies in a glaucoma animal model

Although elevated intraocular pressure (IOP) remains the major risk factor in glaucoma, neurodegenerative processes continue despite effective IOP lowering. Altered α-synuclein antibody (Abs) levels have been reported to play a crucial role. This study aimed at identifying whether α-synuclein Abs are capable to decelerate neuronal decay while providing insights into proteomic changes. Four groups of Sprague Dawley rats received episcleral vein occlusion: (1) CTRL, no intravitreal injection, n = 6, (2) CTRL IgG, intravitreal injection of unspecific IgG, n = 5, (3) Buffer, intravitreal injection of buffer, n = 6, (4), α-synuclein Ab, intravitreal injection of α-synuclein Ab, n = 5. IOP and retinal nerve fiber layer thickness (RNFLT) were monitored and immunohistochemistry, microarray and proteomic analysis were performed. RNFLT was reduced in CTRL, CTRL IgG and Buffer group (all p < 0.01) and α-synuclein Ab group (p = 0.17). Axon and RGC density showed an increased neurodegeneration in CTRL, CTRL IgG and Buffer group (all p < 0.01) and increased neuronal survival in α-synuclein Ab group (p = 0.38 and 0.06, respectively) compared with fellow eyes. Proteomic analysis revealed alterations of cofilin 1 and superoxide dismutase 1 expression. This data indicate that α-synuclein Ab might indirectly modulate the actin cytoskeleton organization and negatively regulate apoptotic processes via cofilin 1 and superoxide dismutase 1.

A Bioengineering Approach to Myopia Control Tested in a Guinea Pig Model.

PurposeTo investigate the biocompatibility of an injectable hydrogel and its ability to control myopia progression in guinea pigs.MethodsThe study used a hydrogel synthesized from acrylated hyaluronic acid with a conjugated cell-binding peptide and enzymatically degradable crosslinker. Seven-day-old guinea pigs were first form deprived (FD) with diffusers for 1 week. One group was kept as an FD-only control; two groups received a sub-Tenon's capsule injection of either hydrogel or buffer (sham surgery) at the posterior pole of the eye. Form deprivation treatments were then continued for 3 additional weeks. Treatment effects were evaluated in terms of ocular axial length and refractive error. Safety was evaluated via intraocular pressure (IOP), visual acuity, flash electroretinograms (ERG), and histology.ResultsBoth hydrogel and sham surgery groups showed significantly reduced axial elongation and myopia progression compared to the FD-only group. For axial lengths, net changes in interocular difference (treated minus control) were 0.04 ± 0.06, 0.02 ± 0.09, and 0.24 ± 0.08 mm for hydrogel, sham, and FD-only groups, respectively (P = 0.0006). Intraocular pressures, visual acuities, and ERGs of treated eyes were not significantly different from contralateral controls. Extensive cell migration into the implants was evident. Both surgery groups showed noticeable Tenon's capsule thickening.ConclusionsSub-Tenon's capsule injections of both hydrogel and buffer inhibited myopia progression, with no adverse effects on ocular health. The latter unexpected effect warrants further investigation as a potential novel myopia control therapy. That the hydrogel implant supported significant cell infiltration offers further proof of its biocompatibility, with potential application as a tool for drug and cell delivery.

Bone Repair Process in Defects of Diabetic Rats Filled with Autogenous Bone Graft and Covered with Homogenous Bone Matrix Membrane or Polytetrafluoroethylene Membrane.

To analyze the process of repair of bone defects in diabetic rats filled with autogenous bone and covered with membranes of homogenous bone matrix or expanded polytetrafluoroethylene (ePTFE). One hundred twenty male rats were divided into two groups: group 1 (IC), without systemic alterations (control), received an intravenous injection of citrate buffer at 0.01 M, pH 4.5; group 2 (IID) (diabetic) received an intravenous injection of streptozotocin (Sigma-Aldrich) dissolved in 0.01 M citrate buffer (pH 4.5) at a concentration of 35 mg/kg. After glycemic control was achieved, the rats were subdivided into three groups: SM (surgical cavity of the left tibia filled with autogenous bone graft, not covered by membrane); MH (surgical cavity filled with autogenous bone graft and covered with homogenous membrane); and MX (surgical cavity filled with autogenous bone graft and covered with synthetic ePTFE membrane). At 10 and 60 days, the defects in the tibiae were analyzed histologically and histometrically. At 10 days, no statistically significant differences were found between the groups. However, the bone tissue of the diabetic group was qualitatively worse than that of the control group. At 60 days, a delay was found in the bone-repair process in wounds covered by the membranes regardless of the systemic state, but the quality of the newly formed bone in the wounds covered by the membranes was better in both groups. At 60 days, the diabetic group treated with homogenous membrane experienced less bone formation when compared with the nondiabetic group, and this difference was statistically significant. Such differences were even greater between the groups treated without the membrane (P < .01). The homogenous membrane exhibited excellent biocompatibility and was incorporated into the newly formed bone in later periods, both in diabetic and nondiabetic rats.

Kinetic Binding Analysis of Biomolecular Interactions Using Surface Plasmon Resonance System

Surface plasmon resonance (SPR) technology is a versatile and sensitive tool widely used for kinetic studies of interactions between unlabelled biomolecules in real time. The interactions of 4-carboxy benzene sulfonamide (4-CBS) inhibitor, with the enzyme carbonic anhydrase II (CA II) and Interleukin 2 with its antibody (IL 2 antibody) were determined at different concentrations by injecting at a flow rate of 100 μl.min-1 for 60s (100 μl) for a 1 min association phase, which was followed by a 2 min dissociation phase. Langmuir model yielded the rate constants ka and kd , which were of the order of 1.58E+3 M-1. s-1 and 0.04 s-1, respectively, and equilibrium dissociation constant was 2.45E-7 M. Automatic artifact removal, using software options and double referencing (by injection of running buffer to nullify non-specific interactions), further improved the quality of response. Langmuir model yielded the rate constants ka and kd , which were of the order of 2.79E+5 M-1.s-1 and 1.43E-4 s-1, respectively, and equilibrium dissociation constant was 5.17E-7 M. The results illustrate the potential of such kinetic analysis in disease management, drug discovery and for detection of pathogens, toxins, pesticides and antibiotic residues based on either enzyme inhibition or antigen-antibody binding

Effects of propranolol on oxygen-induced retinal neovascularization in mouse

To investigate whether propranolol application as collyrium or intraperitoneal (IP) injection can promote the recovery of oxygen-induced retinopathy (OIR). Thirty-six 7-day-old mice were divided into the following 6 groups: normal control, propranolol eye drops, propranolol IP injection, eye drops negative control, IP injection negative control, and pathological model with 6 mice in each. In a typical model of OIR, litters of mice pups with their nursing mothers were exposed to an infant incubator to high oxygen concentration (75 ± 5)% between postnatal day (PD) 7 and PD12, prior to returning to room air. Two routes of propranolol treatment were assessed from PD12 to PD17: IP injection and eye drop, with doses 2 mg/(kg·time), three times a day. Another three groups were given citric acid buffer eye drops, IP injection of citric acid buffer, and negative control were not treated with any drug. Neonatal mice fed in normal conditions served as normal control. Mice were sacrificed at PD17 to evaluate the morphological changes of retinal vessels by fluorescein isothiocyanate-dextran perfusion and retinal whole mount. The retinal neovascularization was evaluated by counting the number of nuclei of the endothelial cell breaking through the internal limiting membrane (ILM). Compared with the oxygen-exposed group, the branches of retinal vessels went normal with a less un-perfused area in the propranolol eye drops and propranolol IP injection groups [(38.9 ± 9.9)% and (5.6 ± 2.3)% vs. (16.2 ± 10.0)% and (2.2 ± 0.8)%, (25.9 ± 5.0)% and (2.1 ± 2.7)%, F=36.12 and 14.55, P both<0.001]. The number of nuclei of endothelial cells breaking through the ILM on the retinal cross-section in the propranolol eye drops group decreased (14.2 ± 5.1) per slide, which was less than that in the oxygen-exposed group (49.1 ± 8.9) per slide and the propranolol IP injection group (18.0 ± 5.9) per slide; it was also less than that in the eye drops negative control group (47.4 ± 8.1) per slide (F=187.60, P<0.05). Moreover, the number of nuclei of endothelial cells breaking through the ILM on the retinal cross-section in the propranolol IP injection group was less than that in the IP injection negative control group (49.9 ± 7.1) per slide (P<0.05). Propranolol could effectively inhibit the formation of retinal neovascularization in mice; the eye drops was more effective than the IP injection.

Imaging Cellular Inorganic Phosphate in Caenorhabditis elegans Using a Genetically Encoded FRET-Based Biosensor.

Inorganic phosphate (Pi) has central roles in metabolism, cell signaling and energy conversion. The distribution of Pi to each cell and cellular compartment of an animal must be tightly coordinated with its dietary supply and with the varied metabolic demands of individual cells. An analytical method for monitoring Pi dynamics with spatial and temporal resolution is therefore needed to gain a comprehensive understanding of mechanisms governing the transport and recycling of this essential nutrient. Here we demonstrate the utility of a genetically encoded FRET-based Pi sensor to assess cellular Pi levels in the nematode Caenorhabditis elegans. The sensor was expressed in different cells and tissues of the animal, including head neurons, tail neurons, pharyngeal muscle, and the intestine. Cytosolic Pi concentrations were monitored using ratiometric imaging. Injection of phosphate buffer into intestinal cells confirmed that the sensor was responsive to changes in Pi concentration in vivo. Live Pi imaging revealed cell-specific and developmental stage-specific differences in cytosolic Pi concentrations. In addition, cellular Pi levels were perturbed by food deprivation and by exposure to the respiratory inhibitor cyanide. These results suggest that Pi concentration is a sensitive indicator of metabolic status. Moreover, we propose that live Pi imaging in C. elegans is a powerful approach to discern mechanisms that govern Pi distribution in individual cells and throughout an animal.

Effects of FoxO1 on podocyte injury in diabetic rats

ObjectiveThis study was designed to investigate the protective effect of forkhead transcription factor O1 (FoxO1) on podocyte injury in rats with diabetic nephropathy. MethodsStreptozotocin-induced diabetic rats were served as DM group, while DM rats transfected with blank lentiviral vectors (LV-pSC-GFP) or lentiviral vectors carrying constitutively active FoxO1 (LV-CA-FoxO1) were served as LV-NC group or LV-CA group, respectively. The control group (NG) consisted of uninduced rats that received an injection of diluent buffer. At 2, 4, and 8 weeks after transfection, the levels of urine albumin, blood glucose, blood urea nitrogen, serum creatinine and urine podocalyxin were measured. Real-time PCR and western blotting were performed to measure mRNA and protein levels of FoxO1, podocalyxin, nephrin, and desmin in renal cortex. In addition, light and electron microscopy were used to detect structural changes in the glomerulus and podocytes. ResultsCompared with the rats in LV-NC and DM groups, LV-CA rats showed a significant increase in FoxO1 mRNA and protein levels and a distinct decrease in urine albumin, blood urea nitrogen, and serum creatinine (except at the two-week time point) levels (p < 0.05). Podocalyxin and nephrin mRNA and protein levels increased (p < 0.05), whereas desmin mRNA and protein levels decreased (p < 0.05). Pathological changes in glomerulus were also ameliorated in LV-CA group. ConclusionsUpregulating expression of FoxO1 by transduction with recombinant lentivirus ameliorates podocyte injury in diabetic rats.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

Effects of forkhead transcription factor O1 on the expression of type IV collagen and desmin in podocytes of diabetic rats

To study the effects of forkhead transcription factor O1(FoxO1)on the expression of type Ⅳ collagen and desmin in podocytes of diabetic rats. Streptozotocin-induced diabetic rats were divided into three groups: diabetic rats(DM group), rats transfected with blank lentiviral vectors(diabetes mellitus plus LV-pSC-GFP group, LV-NC group), and rats which were transfected with lentiviral vectors carrying constitutively active FoxO1(diabetes mellitus plus LV-FoxO1-AAA group, LV-CA group). Rats which received an injection of diluent buffer served as normal control. At 2, 4, and 8 weeks after transfection, the levels of urine albumin, blood glucose, blood urea nitrogen, and serum creatinine were measured. Realtime PCR and Western blotting were performed to measure the mRNA and protein levels of FoxO1, COL4A3, COL4A5, and desmin in the renal cortex. Moreover, light microscope and electron microscope were used to observe the structural changes in glomerulus and podocytes. Compared with LV-NC and DM group, in LV-CA group, there was a significant increase in the mRNA and protein levels of FoxO1, and a distinct decrease in the levels of urine albumin, blood urea nitrogen and serum creatinine of rats(except at the two-week time point)(all P<0.05), the mRNA and protein levels of COL4A3, COL4A5, and desmin were all decreased(all P<0.05), and pathological changes in kidney were also improved. Upregulating the expression of FoxO1 by transfecting with constructed lentiviral vectors can definitely improve the abnormal expression of type Ⅳ collagen and desmin in podocytes of diabetic rats. (Chin J Endocrinol Metab, 2015, 31: 354-359) Key words: Diabetic nephropathy; Forkhead transcription factor O1; Podocytes; Type Ⅳ collagen; Desmin

Using induced pluripotent stem cell-derived conditional medium to attenuate the light-induced photodamaged retina of rats

BackgroundLight injury to photoreceptor cells and retinal pigment epithelium may lead to oxidative stress and irreversible degeneration of retina, especially degeneration of the high energy-demanded macula. The model of retinal photodamage could be applied to age-related macular degeneration and other degenerative retinal diseases for exploring new treatments. Based on broadly investigated induced pluripotent stem cells (iPSC) in the field of retinal degeneration, we aimed to clarify further how the interaction progresses between iPSC-conditional medium (CM) and light-damaged retina. MethodsiPSCs were generated from murine embryonic fibroblasts of C57/B6 mice by retroviral transfection of three factors: Oct4, Sox2, and Klf4. Cytokine array was performed to analyze the components of CM. Sprague–Dawley rats receiving white light exposure to retina were viewed as an animal model of light injury. The rats were divided into four subgroups: light-injured rats receiving intravitreal injection of iPSC-CM, apoptotic iPSC-CM, or sodium phosphate buffer (PBS); and a control group without light damage. The electroretinography and thickness of outer nuclear layer were measured to document the therapeutic effects in each condition. Apoptosis arrays for detecting annexin V and caspase 3 were performed in the retinal tissues from each group. ResultsMurine embryonic fibroblasts were induced into iPSCs and expressed the marker genes similar to embryonic stem cells. These iPSCs can differentiate into Embryoid bodies (EBs), three germ layers in vitro and develop teratoma in severe combined immunodeficiency mice. The quantitative polymerase chain reaction of our iPSC-CM showed significantly elevated fibroblast growth factor-2, glial cell-derived neurotrophic factor, and insulin-like growth factor-binding proteins-1, -2, and -3. Compared to rats without photodamage, the light-injured rats receiving iPSC-CM had less reduction of outer nuclear layer thickness on Day 21 than other groups treated with either PBS or apoptotic iPSC-CM. In the same animal model, both a- and b-waves of electroretinography measurement in the group treated with iPSC-CM were significantly maintained compared to the control group and others with apoptotic iPSC-CM or PBS treatment. The apoptosis assay also demonstrated lower levels of annexin V and caspase 3 in the group with iPSC-CM treatment than in other groups presenting increasing apoptotic markers. ConclusionThe conditional medium of iPSCs contains plenty of cytoprotective, immune-modulative and rescue chemicals, contributing to the maintenance of neuronal function and retinal layers in light-damaged retina compared with apoptotic iPSC-CM and PBS. The antiapoptotic effect of iPSC-CM also shows promise in restoring damaged neurons. This result demonstrates that iPSC-CM may serve as an alternative to cell therapy alone to treat retinal light damage and maintain functional and structural integrity of the retina.

Changes of serum osteocalcin, calcium, and potassium in a rat model of type 2 diabetes.

Changes in serum levels of calcium (Ca), phosphorus (P), Ca phosphate, alkaline phosphatase (AKP), and osteocalcin (BGP) were assessed in a model of type 2 diabetes in 8-weeks-old Sprague-Dawley rats. Throughout the study, the animals in Group A consumed high-carbohydrate, high-fat diet and were administered two intraperitoneal injections of streptozocin (STZ) at 4 (25 mg/kg) and 8 (40 mg/kg) weeks after diet initiation. Animals in Group B received similar diet but intraperitoneal injections with citrate buffer. The control group rats (Group C) were fed normal diet and received citrate buffer injections. The Ca levels in Group A were significantly decreased at 12 and 16 weeks, while P levels were significantly lower at 8, 12, and 16 weeks after the first STZ injection (p < 0.05 vs. Groups B and C). The Ca phosphate levels in Group A were significantly lower at 16 weeks (p < 0.05 vs. Groups B and C). There was a discrepancy in kinetics of AKP and BGP levels in Group A, such that the former showed a significant surge at 8 weeks (p < 0.05 vs. Groups B and C) followed by a decline at 16 weeks (p < 0.05 vs. Groups B and C), while the levels of the latter consistently decreased and became significantly decreased at 12 and 16 weeks (p < 0.05 vs. Groups B and C). In conclusion, these findings may lead to better understanding of the pathogenesis of osteoporosis in diabetic rats.

Stem cells with FGF4-bFGF fused gene enhances the expression of bFGF and improves myocardial repair in rats

The aim of this study was to investigate whether the modification of bone marrow-derived mesenchymal stem cells (BMSCs) with the fused FGF4 (fibroblast growth factor 4)-bFGF (basic fibroblast growth factor) gene could improve the expression and secretion of BFGF, and increase the efficacies in repairing infarcted myocardium. We used In-Fusion technique to construct recombinant lentiviral vectors containing the individual gene of bFGF, enhanced green fluorescent protein (EGFP), or genes of FGF4-bFGF and EGFP, and then transfected these lentiviruses into rat BMSCs. We conducted an in vitro experiment to compare the secretion of bFGF in BMSCs infected by these lentiviruses and also examined their therapeutic effects in the treatment of myocardial infraction in a rodent study. Sixty rats were tested in the following five conditions: Group-SHAM received only sham operation as controls; Group-AMI received only injection of placebo PBS buffer; Group-BMSC, Group-bFGF and Group-FGF4-bFGF received implantation of BMSCs with empty lentivirus, bFGF lentivirus, and FGF4-bFGF lentivirus, respectively. Our results found out that the transplanted FGF4-bFGF BMSCs had the highest survival rate, and also the highest myocardial expression of bFGF and microvascular density as evidenced by Western blotting and immunohistochemistry, respectively. As compared to other groups, the Group-FGF4-BFGF rats had the lowest myocardial fibrotic fraction, and the highest left ventricular ejection fraction. These results suggest that the modification of BMSCs with the FGF4-bFGF fused gene can not only increase the expression of bFGF but also improve its secretion. The FGF4-bFGF BMSCs thus can enhance the survival of the transplanted cells, diminish myocardial fibrosis, promote myocardial angiogenesis, and improve cardiac functions.

Protective effects of curcumin on retinal Müller cell in early diabetic rats.

To explore the effects and potential mechanisms of curcumin on retinal Müller cell in early diabetic rats. Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ). Male Sprague-Dawley (SD) rats were randomly assigned into 4 groups: control group (naïve SD rats administered with a single intraperitoneal injection of citric buffer), diabetic group (STZ-diabetic rats), dimethyl sulfoxide (DMSO) group (diabetic rats intraperitoneally administered with mixture of DMSO and normal saline, once a day) and curcumin group (diabetic rats intraperitoneally administered with curcumin, 80mg/kg, once a day). Three months after diabetes onset, malondialdehyde (MDA, indication of oxidative stress level) and reduced glutathione (GSH) in retina were detected with kits, glial fibrillary acidic protein (GFAP) in retina was revealed by immunohistochemistry and Western blot, and retinal glutamine synthetase (GS) were observed by Western blot. Compared with control group, retinal MDA was increased, and GSH was decreased in diabetic and DMSO groups (P<0.05, respectively). While, retinal MDA and GSH in curcumin group showed no difference compared with control group (P>0.05). Furthermore, up-regulation of retinal GFAP and down-regulation of retinal GS were detected in diabetic and DMSO groups, and no alteration could be observed in curcumin group revealed with Western blot. Compared with control group, retinal Müller cells showed significant increase in GFAP immunochemistry staining in diabetic and DMSO groups. Moreover, GFAP-positive staining was decreased in curcumin group compared with diabetic group. Curcumin inhibits diabetic retinal oxidative stress, protects Müller cell, and prevents the down-regulation of GS in diabetic retina. Therefore, curcumin has a therapeutic potential in the treatment of diabetic retinopathy (DR).

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in-capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β-nicotinamide adenine dinucleotide 2'-phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β-nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver-Burk equation, the Michaelis-Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.

Prestorage in ovo injection of biological buffers: An approach to improve hatchability in long-term stored eggs

A hypothesis was tested that the in ovo injection of biological buffers may reinforce the buffering capacity of albumen, thereby withstanding the increase in albumen pH during storage and improving hatchability and chick quality in long-term stored eggs. Hatching eggs (n = 2,420) were randomly assigned to 11 treatment groups (4 replicates of 55 eggs each) and injected (d 1) with distilled water, 25 or 50 mM HEPES (H25 and H50), Bicine (B25 and B50), Tris (T25 and T50), and Bis-Tris-propane (BTP25 and BTP50) solutions or were not injected (intact: control; or pricked with a needle: N). The eggs were then stored for 14 d during which the egg internal characteristics were evaluated at 1, 2, 3, 4, 5, 8, and 13 d of storage (n = 924 in total) and the remaining eggs (n = 1,496) were incubated. A decrease in albumen pH was found for H25, H50, B50, and BTP25 groups from 2 through 5 d postinjection. Eggs receiving H25, H50, and B50 recorded a higher albumen index (at 13 d of storage) and Haugh unit (between 8 and 13 d of storage) compared with the control. Interestingly, the hatchability of fertile eggs was influenced by the treatment effect (P = 0.0001) where the eggs receiving H25 (88.3%), H50 (88.9%), B50 (88.4%), and BTP25 (87.6%) recorded higher values than that of control (82.1%), associated with a decreased early embryonic mortality rate (P < 0.0001). In ovo injection of Tris buffer, however, profoundly decreased the hatchability (47.2 and 29.0% for T25 and T50, respectively) and percentage of first-grade chicks (67.5 and 63.6% for T25 and T50, respectively) compared with the control (90.1%). In conclusion, prestorage in ovo injection of H25, H50, B50, and BTP25 improved hatchability in long-term stored eggs in which a decreased albumen pH during the d 2 through 5 of storage period might be involved.

Trigonelline ameliorates diabetic hypertensive nephropathy by suppression of oxidative stress in kidney and reduction in renal cell apoptosis and fibrosis in streptozotocin induced neonatal diabetic (nSTZ) rats

Oxidative stress and apoptotic cell death in kidney have been suggested as contributing factors in the development and complication of diabetes especially in diabetic nephropathy (DN). This study investigated the effects of trigonelline (TG) on the renal functional, morphological changes and renal apoptosis in neonatal diabetic rats, a model of non-insulin-dependent diabetes mellitus. Diabetes mellitus was induced in one day old neonatal Wistar rat pups by an intraperitoneal (i.p.) injection of streptozotocin (STZ) (50mg/kg) and monitored for 16weeks thereafter. The diabetic rats were divided as follows: the nSTZ diabetic group, the TG (50mg/kg) treated diabetic group, and the TG (100mg/kg) treated diabetic group. The age matched nondiabetic group received an injection of citrate buffer (0.1M, pH4.5). At the end kidney samples were taken for light microscopic examinations. The levels of serum creatinine and BUN were significantly low in TG (100mg/kg) treated diabetic rats. Glomerular filtration rate was improved in TG treated rats. The activities of antioxidant enzyme and membrane bound enzyme were decreased and the levels of tumor necrotic factor (TNF-α) and hydroxyproline content were increased in renal tissues of the diabetic group. TG (100mg/kg/day) treatment for a period of 4weeks showed significant ameliorative effects on all the biochemical parameters studied. Biochemical findings were supported by histological studies. The degenerative changes in kidney tissue and fibrosis were alleviated in the TG treated groups. These results suggested that TG might have a significant role in alleviating kidney damage in nSTZ-diabetic rats.

Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

Normal epidermal growth factor receptor signaling is dispensable for bone anabolic effects of parathyroid hormone

Although the bone anabolic properties of intermittent parathyroid hormone (PTH) have long been employed in the treatment of osteoporosis, the molecular mechanisms behind this action remain largely unknown. Previous studies showed that PTH increases the expression and the activity of epidermal growth factor receptor (EGFR) in osteoblasts, and activation of ERK1/2 by PTH in osteoblasts was demonstrated to induce the proteolytical release of EGFR ligands and EGFR transactivation. However, conclusive evidence for an important role of the EGFR system in mediating the anabolic actions of intermittent PTH on bone in vivo is lacking. Here, we evaluated the effects of intermittent PTH on bone in Waved-5 (Wa5) mice which carry an antimorphic Egfr allele whose product acts as a dominant negative receptor. Heterozygous Wa5 females and control littermates received a subcutaneous injection of PTH (80 μg/kg) or buffer on 5 days per week for 4 weeks. Wa5 mice had slightly lower total bone mineral density (BMD), but normal cancellous bone volume and turnover in the distal femoral metaphysis. The presence of the antimorphic Egfr allele neither influenced the PTH-induced increase in serum osteocalcin nor the increases in distal femoral BMD, cortical thickness, cancellous bone volume, and cancellous bone formation rate. Similarly, the PTH-induced rise in lumbar vertebral BMD was unchanged in Wa5 relative to wild-type mice. Wa5-derived osteoblasts showed considerably lower basal extracellular signal-regulated kinase 1/2 (ERK1/2) activation as compared to control osteoblasts. Whereas activation of ERK1/2 by the EGFR ligand amphiregulin was largely blocked in Wa5 osteoblasts, treatment with PTH induced ERK1/2 activation comparable to that observed in control osteoblasts, relative to baseline levels. Our data indicate that impairment of EGFR signaling does not affect the anabolic action of intermittent PTH on cancellous and cortical bone.

Resveratrol blocks diabetes‐induced early vascular lesions and vascular endothelial growth factor induction in mouse retinas

Vessel leakage and loss of pericytes are early signs of diabetic retinopathy (DR), which leads to vision loss. Upregulation of the vascular endothelial growth factor (VEGF) during diabetes plays a key role in mediating these vascular lesions. The aim of this study is to investigate the effects of resveratrol, a natural plant-derived phytoalexin, on vascular damage and VEGF induction in mouse retinas of early diabetes. Diabetes was induced in C57BL/6 mice by five consecutive-intraperitoneal injections of 55 mg/kg of streptozotocin (STZ). Animals injected with buffer only were used as controls. Beginning 1 month after the fifth injection of STZ or buffer, 20 mg/kg of resveratrol was administered by oral gavage daily for 4 weeks to diabetic and control mice, and all mice were killed 2 months after the injections. We assessed vessel leakage, pericyte loss and VEGF protein expression in mouse retinas of 2-month diabetes compared with controls with or without resveratrol treatment. Diabetes led to increase vessel leakage, pericyte loss and VEGF protein level in the mouse retinas compared with controls; however, these changes were effectively blocked by resveratrol treatment. Our data suggest that resveratrol is effective to decrease vascular lesions and VEGF induction in mouse retinas of early diabetes.