The Effects of Calpain Inhibition on IkBα Degradation After Activation of PBMCs: Identification of the Calpain Cleavage Sites

Human peripheral blood mononuclear cells (PBMCs) were activated using anti-CD3/CD28 (HIT3A/CD28.2) resulting in degradation of IkB alpha, an inhibitor of NFkB, relative to unactivated cells. Degradation of IkB alpha began by 30 min and proceeded for at least 5 h. Calpeptin, a calpain inhibitor, inhibited IkB alpha degradation in a time- and dose-dependent manner. Furthermore, calpain inhibition increased IkB alpha levels compared to nonactivated controls. Recombinant IkB alpha was incubated with purified porcine m-calpain in the presence of 0.1% Triton X-100, and the degradation products were monitored by SDS-PAGE and sequenced. Most of the degradation products were peptides derived from calpain, but one was derived from IkB alpha cleaved between amino acids 50 and 51 (glutamine and glutamic acid). The liberated fragment included the entire signal response domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. The results suggest that calpain plays an important role in IkB alpha degradation, a crucial event in T cell activation.     

In vitro and in vivo antioxidant activity of exopolysaccharide fractions from Bifidobacterium animalis RH

The aim of the study was to purify the exopolysaccharides (EPS) produced from Bifidobacterium animalis RH, which was isolated from the feces of Bama centenarians in Guangxi of China, and evaluate their antioxidant activities in vitro and in vivo. 2 fractions, a neutral EPS fraction (EPSa) and an acidic EPS fraction (EPSb), were obtained and compared for antioxidative activity. In vitro, they both showed remarkable inhibition effect on lipid peroxidation and strong DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, in which the last two were measured by the electron spin resonance (ESR). In vivo, EPSa and EPSb were orally administrated for 30 days in a d-galactose induced aged mice model. As results, they both could significantly increase the activities of SOD, CAT and total antioxidant capacity (TAOC) in serums and glutathione GST in livers. They also could inhibit significantly the formation of MDA in serums and livers, and reduce the activity of MAO and lipofuscin accumulation in mice brain. Moreover, EPSb exhibited much higher antioxidant activities than EPSa in vitro and in vivo. The results suggested that EPS fractions of Bifidobacterium animalis RH had direct and potent antioxidant activities.     

Hexarelin Protects Rodent Pancreatic Β-Cells Function from Cytotoxic Effects of Streptozotocin Involving Mitochondrial Signalling Pathways In Vivo and In Vitro

Mitochondrial functions are crucial for pancreatic β-cell survival and glucose-induced insulin secretion. Hexarelin (Hex) is a synthetic small peptide ghrelin analogue, which has been shown to protect cardiomyocytes from the ischemia-reperfusion process. In this study, we used in vitro and in vivo models of streptozotocin (STZ)-induced β-cell damage to study the protective effect of Hex and the associated mechanisms. We found that STZ produced a cytotoxic effect in a dose- and time-dependent manner in MIN6 cells (a mouse β-cell line). Hex (1.0 μM) decreased the STZ-induced damage in β-cells. Rhodamine 123 assay and superoxide DHE production assay revealed that Hex ameliorated STZ-induced mitochondrial damage and excessive superoxide activity in β-cells. In addition, Hex significantly reduced STZ-induced expression of cleaved Caspases-3, Caspases-9 and the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 in MIN6 cells. We further examined the in vivo effect of Hex in a rat model of type 1 diabetes induced by STZ injection. Hex ameliorated STZ-induced decrease in plasma insulin and protected the structure of islets from STZ-induced disruption. Hex also ameliorated STZ-induced expression of cleaved Caspase-9 and the Bax in β-cells. In conclusion, our data indicate that Hex is able to protects β-cell mass from STZ-caused cytotoxic effects involving mitochondrial pathways in vitro and in vivo. Hex may serve as a potential protective agent for the management of diabetes.     

In vivo Neuroprotective Activity of Epopeptide AB Against Ischemic Damage

Erythropoietin (Epo) is a hematopoietic factor, which stimulates proliferation and differentiation of erythroid precursor cells. Epo also functions as a neuroprotective factor and protects neurons from ischemic damage. Recently a 17-mer peptide sequence (Epopeptide AB) in Epo (AEHCSLNENITVPDTKV) with a neuroprotective function was reported. In this study, we showed in vivo evidence that Epopeptide AB protected neurons from ischemic damage at similar dose compared to Epo. Epopeptide AB could not stimulate the proliferation of Epo-dependent growing murine myeloid Ep-FDC-P2 cells and also did not compete the proliferative function of Epo on these cells. Together with these results, Epopeptide AB did not transduce signals through direct binding to the known Epo receptor on hematopoietic cells but has neuroprotective activity against ischemia. These authors contributed equally to this paper     

In vivo response to the different LET regions of a 55 MeV helium ion beam

We evaluated the in vivo response to heavy particle irradiation in rat tail epidermis using silver-stained nucleolar organizer regions (AgNOR) as the end-point. The energy degradation of the beam across the circular section of the tail allowed us to study the damage elicited by two different LET regions of a helium beam, i.e. non-Bragg peak (NBP) and Bragg peak (BP), at different sites on the same sample. The tails were locally irradiated with a helium ion beam at different fluences. AgNOR exhibited marked variations between tissue areas only a few micrometers apart within each tail exposed to a given beam fluence. An analysis of the AgNOR variations in NBP and BP areas of tails exposed to different beam fluences revealed a dose-dependent effect. The AgNOR provide quantitative evidence of differential damage in neighboring tissue areas exposed to different LET regions of a helium-ion beam.     

Decreased Succinate Dehydrogenase Activity of Gamma and Alpha Motoneurons in Mouse Spinal Cords Following 13 Weeks of Exposure to Microgravity

Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm²) gamma and medium-sized (300–700 μm²) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm²) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential

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Effects of a lipid environment on the fibrillogenic pathway of the N-terminal polypeptide of human apolipoprotein A-I, responsible for in vivo amyloid fibril formation

In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an α-helical state, which undergoes aggregation at 37°C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of α-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.     

“In vivo” pH values of the estrous reproductive tract of the gilt

The hydrogen ion concentration of the fluids of various portions of the reproductive tract was determined in 6 estrous gilts. Determinations were made with a pH cardiovascular catheter electrode placed in the lumen of the oviduct, uterus, cervix and vagina and a reference electrode placed in the peritoneal cavity. The mean pH values were 7.02, 6.98, 7.49, and 6.61 for the oviduct, uterus, cervix and vagina, respectively.     

In vivo and in vitro function of human UDP-galactose 4'-epimerase variants

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4′-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4′-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4′-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.     

Reconstruction of the Human Colon Epithelium In Vivo

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In vitro and in vivo antifungal activates of the essential oils of various plants against strawberry grey mould disease agent Botrytis cinerea

In order to investigate the effects of antifungal essential oils on postharvest decay and some quality factors of strawberry fruit, experiments were conducted under in vitro and in vivo conditions. The antifungal activates of essential oils obtained from fennel, anis, peppermint and cinnamon at concentrations 0, 200, 400, 600 and 800 μL L^?1 were investigated against Botrytis cinerea with four replications. In vitro results showed that the growth of B. cinerea was completely inhibited by fennel, cinnamon and anis essential oils at relatively low concentrations (400–800 μL L^?1). In vivo results showed that all the used essential oils at all applied concentrations caused an increase in the shelf life and inhibited of B. cinerea growth on strawberry fruits completely in comparison to the controls. The results of this study confirmed the antifungal effect of four essential oils in both in vitro and on fruit postharvest.     

Effects In Vitro of Guanidinoacetate on Adenine Nucleotide Hydrolysis and Acetylcholinesterase Activity in Tissues from Adult Rats

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. The aim of this work was to investigate the in vitro effect of guanidinoacetate in NTPDase, 5′-nucleotidase and acetylcholinesterase activities in the synaptosomes, platelets and blood of rats. The results showed that in synaptosomes the NTPDase and 5′-nucleotidase activities were inhibited significantly in the presence of GAA at concentrations of 50, 100, 150 and 200 μM (P < 0.05). However, in platelets GAA at the same concentrations caused a significant increase in the activities of these two enzymes (P < 0.05). In relation to the acetylcholinesterase activity, GAA caused a significant inhibition in the activity of this enzyme in blood at concentrations of 150 and 200 μM (P < 0.05), but did not alter the acetylcholinesterase activity in synaptosomes from the cerebral cortex. Our results suggest that alterations caused by GAA in the activities of these enzymes may contribute to the understanding of the neurological dysfunction of GAMT-deficient patients.     

Pharmacological Inhibition of Necroptosis Protects from Dopaminergic Neuronal Cell Death in Parkinson’s Disease Models

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Targeted Inhibition of Phospholipase C γ2 Adaptor Function Blocks Osteoclastogenesis and Protects from Pathological Osteolysis

Phospholipase C γ2 (PLCγ2) is a critical regulator of innate immune cells and osteoclasts (OCs) during inflammatory arthritis. Both the catalytic domain and the adaptor motifs of PLCγ2 are required for OC formation and function. Due to the high homology between the catalytic domains of PLCγ2 and the ubiquitously expressed PLCγ1, molecules encompassing the adaptor motifs of PLCγ2 were designed to test the hypothesis that uncoupling the adaptor and catalytic functions of PLCγ2 could specifically inhibit osteoclastogenesis and bone erosion. Wild-type (WT) bone marrow macrophages (BMM) that overexpress the tandem Src homology 2 (SH2) domains of PLCγ2 (SH2(N+C)) failed to form mature OCs and resorb bone in vitro. Activation of the receptor activator of NF-κB (RANK) signaling pathway, which is critical for OC development, was impaired in cells expressing SH2(N+C). Arrest in OC differentiation was evidenced by a reduction of p38 and Iκ-Bα phosphorylation as well as decreased NFATc1 and c-Fos/c-Jun levels. Consistent with our hypothesis, SH2(N+C) abrogated formation of the RANK-Gab2 complex, which mediates NF-κB and AP-1 activation following RANK ligand (RANKL) stimulation. Furthermore, the ability of SH2(N+C) to prevent inflammatory osteolysis was examined in vivo following RANKL or LPS injections over the calvaria. Both models induced osteolysis in the control group, whereas the SH2(N+C)-treated cohort was largely protected from bone erosion. Collectively, these data indicate that inflammatory osteolysis can be abrogated by treatment with a molecule composed of the tandem SH2 domains of PLCγ2.     

Inhibition of Glycogen Synthase Kinase-3β Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

A large number of experimental and clinical studies have confirmed that brief remifentanil exposure can enhance pain sensitivity presenting as opioid-induced hyperalgesia (OIH). N-methyl-D-aspartate (NMDA) receptor antagonists have been reported to inhibit morphine analgesic tolerance in many studies. Recently, we found that glycogen synthase kinase-3β (GSK-3β) modulated NMDA receptor trafficking in a rat model of remifentanil-induced postoperative hyperalgesia. In the current study, it was demonstrated that GSK-3β inhibition prevented remifentanil-induced hyperalgesia via regulating the expression and function of spinal NMDA receptors in vivo and in vitro. We firstly investigated the effects of TDZD-8, a selective GSK-3β inhibitor, on thermal and mechanical hyperalgesia using a rat model of remifentanil-induced hyperalgesia. GSK-3β activity as well as NMDA receptor subunits (NR1, NR2A and NR2B) expression and trafficking in spinal cord L₄-L₅ segments were measured by Western blot analysis. Furthermore, the effects of GSK-3β inhibition on NMDA-induced current amplitude and frequency were studied in spinal cord slices by whole-cell patch-clamp recording. We found that remifentanil infusion at 1 μg·kg^-1·min^-1 and 2 μg·kg^-1·min^-1 caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate of the spinal cord dorsal horn and increased GSK-3β activity in spinal cord dorsal horn. GSK-3β inhibitor TDZD-8 significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was prevented with the application of TDZD-8. These results suggest that inhibition of GSK-3β can significantly ameliorate remifentanil-induced hyperalgesia via modulating the expression and function of NMDA receptors, which present useful insights into the mechanistic action of GSK-3β inhibitor as potential anti-hyperalgesic agents for treating OIH.